Composition and method for vancomycin oral liquid

ABSTRACT

The invention relates to stable vancomycin hydrochloride powder for oral liquid formulations. Also provided herein are methods of using vancomycin oral liquid formulations for the treatment of certain diseases such as Clostridium difficile pseudomembranous colitis and Staphylococcal enterocolitis as well as kits and related products thereof.

RELATED APPLICATIONS

This application is a continuation application which claims benefitunder 35 U.S.C. §120 of U.S. Application Serial No. 16/988,387, filed onAug. 7, 2020, which is a continuation application of 15/126,059, filedSep. 14, 2016, now U.S. Pat. No. 10,959,946, which is a national stagefiling under U.S.C. § 371 of PCT International ApplicationPCT/US2015/020575, filed Mar. 13, 2015, which claims the benefit ofpriority under 35 U.S.C. § 119(e) to U.S. Provisional Application SerialNo. 61/953,076, filed on Mar. 14, 2014, and U.S. Provisional ApplicationSerial No. 61/992,414, filed on May 13, 2014, both entitled “COMPOSITIONAND METHOD FOR VANCOMYCIN ORAL LIQUID”.

BACKGROUND OF THE INVENTION

Clostridium difficile, commonly referred to as C. diff., is a bacteriumbest known for causing antibiotic-associated diarrhea. The bacterium isknown to cause infection when competing bacteria in the gut have beenwiped out following the use of antibiotic treatment. Some peopleinfected with C. diff. are asymptomatic, while others infected mayexperience severe diarrhea, abdominal pain, fever and a distinctive foulstool odor. C. diff. infections are the most common cause ofpseudomembranous colitis and in rare cases can progress to alife-threatening condition such as toxic megacolon. To date, C. diff. isthe most prevalent cause of infectious diarrhea and colitis in theUnited States.

C. diff. was first described in 1935 as Bacillus difficilis followingthe analysis of fecal samples of healthy newborn infants, in a studyconducted by Hall and O′Toole. The species name “difficile” remained dueto the difficulty involved in the isolation and study of these bacteria.C. diff. was later described as an obligate anaerobic, spore-producing,gram-positive rod, belonging to an ancient group of bacteria called theClostridia, giving the name Clostridium difficile. Clostridia are motilebacteria that are especially prevalent in soil, but a commensalbacterium of the human intestine. In 1977, Bartlett identified it as ananaerobic bacterium, potent human pathogen and the etiologic agentresponsible for antibiotic associated pseudomembranous colitis andrecognized it as the major cause of antibiotic-associated diarrhea. C.diff. has since been recognized as a hospital-acquired infection (HAI)pathogen, inflicting morbidity in infected individuals triggered by thewidespread use of the antibiotic clindamycin. Over the following years,the antibiotics in the penicillin and cephalosporin families contributedto the C. diff. epidemic.

C. difficile spores can live outside the body for long periods of time,and can be easily spread from patient to patient in healthcarefacilities. Therefore, hospital patients, nursing home residents andindividuals taking antibiotics are at higher risk for Clostridiumdifficile infection (CDI) due to the increased chance of exposure. Thetransfer of C. diff. occurs via fecal-oral route where the spores passthrough the stomach and ultimately end up in the colon. Once antibioticsare introduced, the normal enteric flora is suppressed making anenvironment in which C. diff can proliferate and produce Clostridiumdifficile-associated diarrhea (CDAD) through toxins A (enterotoxin) andB (cytotoxin). In terms of C. diff., approximately 3% of healthy adultstools test positive where the frequency of stool carriage increases to16 - 35% in hospital patients.

When CDI is suspected, a test varies from institution toinstitution-stool culture, cytotoxin assay, PCR, ELISA, Latexagglutination assay for glutamate dehydrogenase or endoscopy areperformed. The most common test used to diagnose C. diff. is ELISA todetect toxin A and B. Once CDI is diagnosed, treatment is administered.The top three medications prescribed are metronidazole, vancomycin, andfidaxomicin. First-line therapy, for mild to moderate C. difficilediarrhea, is restricted to metronidazole. Second-line therapy for severeinfections and/or treatment failure is vancomycin.Intravenously-administered vancomycin should not be used for thetreatment of C. difficile due to its inability to penetrate the colonand reach therapeutic concentrations via this route of administration.However, orally-administered vancomycin reaches very high concentrationsin the colon (generally 500 - 1000 µg/mL) and cannot be absorbed; thus,oral vancomycin is a favorable choice for the treatment of C. difficilepseudomembranous colitis and Staphylococcal enterocolitis by beingconfined to the site of infection, and is then excreted fecally.

SUMMARY OF THE INVENTION

In some aspects the invention is a non-sterile stable liquid formulationof a compounded solution of vancomycin hydrochloride that is homogenousand stable for at least 30 days at ambient and refrigerated temperatureconditions. In some embodiments the compounded solution of vancomycin iscomprised of a liquid solution that is not Ora-sweet. In someembodiments ambient temperature is 22-28° C. In other embodimentsambient temperature is 25±2° C. In some embodiments the solution ofvancomycin hydrochloride is homogenous and stable under ambienthumidity. For instance, ambient humidity may be 60±5% relative humidity.In other embodiments refrigerated temperature is 2° - 8° C.

In yet other aspects, the invention is a stable powder for compoundingto produce a liquid formulation of a compounded solution of vancomycinhydrochloride, wherein the powder is stable for at least 30 days atambient and refrigerated temperature conditions. In some embodiments thepowder is stable for at least 6 months, 12 months, or 18 months atambient temperatures.

In some embodiments the liquid solution is stable for up to 3 months ataccelerated conditions. In some embodiments accelerated conditions are35-45° C. and 68-78% relative humidity. In other embodiments acceleratedconditions are 40° C. and 74% relative humidity. The liquid solution inyet other embodiments is stable for up to 24 months at ambientconditions.

The liquid solution in some embodiments includes one or more of, or inother embodiments, all of:

-   (a) a buffering agent,-   (b) water,-   (c) a sweetener,-   (d) a flavoring agent,-   (e) a preservative, and-   (f) a dye.

The buffering agent in some embodiments is about 0.12% (w/v) citric acid(anhydrous). In other embodiments the sweetener is about 0.2% (w/v)sucralose. In yet other embodiments the flavoring agent is about 0.05%(w/v) artificial grape flavor. The preservative is about 0.05% (w/v)sodium benzoate in some embodiments. The preservative is about 0.1%(w/v) sodium benzoate in some embodiments. In other embodiments thepreservative is about 0.1% - 0.05% (w/v) sodium benzoate. The dye insome embodiments is about 0.0002% (w/v) D&C Yellow No. 10 and about0.000038% (w/v) FD&C Red No. 40.

The compounded solution of the liquid formulation may be formulated foruse in the treatment of different infectious agents including C.difficile pseudomembranous colitis and Staphylococcal enterocolitis.

In some embodiments the compounded solution is formulated to inhibit thegrowth of bacteria, mold and yeast for at least 30 days at ambient andrefrigerated temperature conditions. In other embodiments the liquidsolution is formulated to inhibit the growth of mold and yeast but notbacteria for greater than 30 days at room temperature conditions.

In other embodiments the concentration of vancomycin in the compoundedsolution is 1-100 mg/ml, 20-60 mg/ml, or 25-50 mg/ml.

A kit is provided in other aspects of the invention. The kit includes afirst container comprising a non-sterile 100% (w/w) vancomycinhydrochloride powder, pre-measured into a respective unit of use amount,a second container comprising an oral liquid solution, pre-measured intoa respective unit of use amount, and instructions for use, wherein thefirst and second containers are of a size such that the vancomycinhydrochloride powder and oral liquid solution can be combined in eitherthe first or second container to produce a compounded solution ofvancomycin hydrochloride, and wherein the compounded solution ofvancomycin hydrochloride is homogenous and stable for at least 30 daysat ambient and refrigerated temperature conditions.

In some embodiments the first or second container is a size to hold a 25mg/mL or 50 mg/mL vancomycin oral liquid solution. In other embodimentsthe first container houses 3.84 g, 7.69 g, 10.76 g, or 15.38 gvancomycin hydrochloride (equivalent to 3.75 g, 7.5 g, 10.5 g, or 15 gvancomycin, respectively). The first or second container may be a sizeto hold a volume appropriate for therapy to treat C. difficilepseudomembranous colitis and/or Staphylococcal enterocolitis; 5 oz. (150mL, as dispensed), 7 oz. (210 mL, as dispensed) and 10 oz. (300 mL, asdispensed).

In certain embodiments, the powder is comprised of vancomycinhydrochloride having microbial assay values of about 900 µg/mg or about1100 µg/mg, compositions having about 85% vancomycin B and about 5% orless any other impurities or other substances and about 4.7%monodechlorovancomycin at the end of a given storage period. The powderdoes not, in some embodiments, contain any additional excipients such asglidants, sweeteners, or flavoring.

Also provided herein are processes for preparing a vancomycin oralliquid formulation. In one aspect, the process comprises the steps of(i) providing a uniform powder comprising 100% (w/w) vancomycinhydrochloride in a bottle; (ii) adding the entire bottle of water,sweeteners, flavoring agents, preservatives, and dye in liquid form(solution component); (iii) shaking the liquid formulation for about 30seconds; and (iv) instructing the patient to shake the bottle wellbefore each use.

A non-sterile stable liquid formulation of a compounded solution ofvancomycin hydrochloride that is homogenous and stable for at least 30days at ambient and refrigerated temperature conditions, wherein thecompounded solution has a high solubility in water and a pH of about2.5-4.5 is provided in other aspects of the invention. In someembodiments the compounded solution is an amber color. In otherembodiments the compounded solution includes a sweetener.

In other aspects, the invention is a non-sterile stable liquidformulation comprising a compounded solution of vancomycin hydrochloridethat has been dissolved in a liquid solution comprising a preservative,wherein the compounded solution is stable for at least 30 days atambient and refrigerated temperature conditions and wherein thepreservative is present in the liquid solution at a concentration rangeof 0.8-0.2% (w/v) .

In another aspect the invention is a liquid solution comprising (a)0.1-0.4% (w/v) citric acid (anhydrous), (b) water, (c) 0.1-0.3% (w/v)sucralose, (d) 0.01-0.1 (w/v) of a flavoring agent, (e) 0.08- 0.02%(w/v) sodium benzoate, and (f) 0.0001-0.0003% (w/v) of a dye. In someembodiments the citric acid is 0.12% (w/v) of the solution. In otherembodiments the sucralose is 0.2% (w/v) of the solution, the flavoringagent is 0.05% (w/v) artificial grape flavor, the sodium benzoate isabout 0.05% (w/v) of the solution and/or the dye comprises about 0.0002%(w/v) D&C Yellow No. 10 and about 0.000038% (w/v) FD&C Red No. 40.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF DRAWINGS

The figures are illustrative only and are not required for enablement ofthe invention disclosed herein.

FIG. 1 illustrates exemplary components of a kit of the invention.

FIG. 2 is a bar graph depicting average MIC values for threeformulations over a 30 day testing period.

FIG. 3 is a graph of dissolution profiles in water for threecommercially available vancomycin products.

FIG. 4 is a graph of dissolution profiles in pH 4.5 buffer for threecommercially available vancomycin products.

FIG. 5 is a graph of dissolution profiles in pH 6.8 buffer for threecommercially available vancomycin products.

FIG. 6 is a graph of dissolution profiles in 0.1 N HCl for threecommercially available vancomycin products.

DETAILED DESCRIPTION

The invention encompasses liquid compounded formulations of Vancomycinhydrochloride powder as well as related compounding kits. The liquidformulations have enhanced stability with respect to available liquidformulations. As is well known in the art compounded formulationsinclude reconstituted formulations.

Also provided herein are methods of treating Clostridium difficilepseudomembranous colitis and Staphylococcal enterocolitis comprisingadministering to a patient, such as a child or an elderly patient, anoral liquid formulation compounded from vancomycin hydrochloride powderas described herein.

Commonly, pediatric and geriatric populations encounter difficulty beingadministered solid oral dosage forms such as capsules. This may lead tonon-compliance with the recommended pharmacotherapy with the solid oraldosage forms and likely results in rendering the therapy ineffective.Solid oral dosage forms are usually not favorable for pediatric andgeriatric populations due to the potential risk of choking.Additionally, certain solid oral dosage forms of medications cannot beadministered simply by crushing (e.g., patients requiring various typesof feeding tubes) because of the coating or drug delivery mechanism bywhich the drug is released. Alternatively, vancomycin hydrochloridepowder for injection is typically mixed with sterile water for injectionas a more cost-effective and accessible option for children, theelderly, and patients with feeding tubes in comparison to the oralcapsules. For most community pharmacies (retail/chain, independent, andthe like), vancomycin hydrochloride powder for injection as well assterile water for injection are not always readily available andexpensive vancomycin hydrochloride oral capsules are usually dispensed.

The current method of overcoming the aforementioned drawbacks of thesolid oral dosage form of vancomycin hydrochloride is mixing multiplevials of vancomycin hydrochloride powder for injection with multiplevials of sterile water for injection to achieve the prescribedconcentration. This method of preparation is cumbersome andtime-consuming for pharmacists in today’s busy pharmacies. Oftentimes,this formulation is not flavored to mask the unpalatable bitterness ofvancomycin hydrochloride; thus, this can significantly lead to adecrease in patient compliance. Additionally, another disadvantage withthe current method of compounding vancomycin oral solution, and withmany compounds, is the potential for contamination. As noted, sterilewater for injection is typically the vehicle for compounding thispreparation and it lacks preservatives. In a study conducted by Malletet al (1982), a precipitate had been documented on day 6 of observationat room temperature in the vancomycin oral solution mixed with distilledwater, which also lacks preservatives. For patients already ill with astaphylococcal or C. difficile infection, any other types of pathogens(e.g., bacteria, viruses, fungi/yeast, and/or mold spores) couldpotentially worsen their intended therapeutic outcome due to theadministration and/or shortening of therapy resulting from thecontaminated product.

The methods and products of the invention overcome the difficultiesarising from prior art methods of administering vancomycinhydrochloride. The compositions provide a number of advantages overconventional oral vancomycin hydrochloride capsules, as well asvancomycin hydrochloride powder for injection vials mixed with sterilewater for injection. For instance, the enhanced stability allows for thepreparation of compounded solutions for longer term administrationprotocols. The compounded solutions also have enhanced accessibility tochildren and the elderly, increased patient compliance to medication,and increased protection against the growth of accidentally introducedyeast and/or mold. The compounded solutions of the invention allow forthe safe and effective oral administration of vancomycin hydrochloridefor the treatment of severe and/or recurrent infections of Clostridiumdifficile pseudomembranous colitis and Staphylococcal enterocolitis.

Vancomycin hydrochloride is commercially available in the form of oralcapsules (e.g., Vancocin®, and also generic) and lyophilized powder forinjection. Vancomycin hydrochloride powder for injection (e.g.,Vancocin®, and also generic), a sterile powder, is typically mixed withsterile water for injection and, occasionally, with other ingredientssuch as sweeteners and/or flavoring agents to be administered as acompounded oral solution. Currently, there is no commercially availablevancomycin hydrochloride powder for oral liquid formulation to providemultiple and flexible (MultiFlex™) dosing which may be used for allpopulations and various indications. The invention provides such acompounded solution.

As used herein, “vancomycin” refers to vancomycin base, its salt, orsolvate or derivative or isomer or polymorph thereof. Suitable compoundsinclude the free base, the organic or inorganic salts, isomers, isomersalts, solvates, polymorphs, complexes, etc. U.S. Pat. No. 4,670,258discloses an exemplary method in the preparation, stability, and storageof vancomycin oral liquid. In some embodiments, the vancomycin used inthe compositions described herein is a vancomycin salt, vancomycinhydrochloride.

The liquid formulations and/or kits described herein may includeadditional ingredients. For instance these additional components mayinclude, but are not limited to, buffering agents, preservatives,sweeteners, flavoring agents, coloring agents. Additional excipientssuch as tonicity agents and chelating agents are within the scope of theembodiments. The compounded solution of the invention is prepared bymixing a powder form of vancomycin hydrochloride with a liquid solution,also referred to as a diluent. The liquid solution or diluent of theinvention imparts improved properties on the compounded solution. It wasdiscovered quite surprisingly that an optimal liquid solution can imparton the compounded solution enhanced stability at room temperaturewithout interfering with the activity of the vancomycin hydrochloride.An example of the optimal liquid solution is presented below:

-   (a) 0.1-0.4% (w/v) citric acid (anhydrous),-   (b) water,-   (c) 0.1-0.3% (w/v) sucralose,-   (d) 0.01-0.1 (w/v) of a flavoring agent,-   (e) 0.08- 0.2% (w/v) sodium benzoate, and-   (f) 0.0001-0.0003% (w/v) of a dye.

Buffering agents maintain the pH when vancomycin hydrochloride powder iscompounded into a liquid form. Non-limiting examples of buffering agentsinclude, but are not limited to, sodium bicarbonate, potassiumbicarbonate, magnesium hydroxide, magnesium lactate, magnesiumgluconate, aluminum hydroxide, aluminum hydroxide/sodium bicarbonateprecipitate, a mixture of an amino acid and a buffer, a mixture ofaluminum glycinate and a buffer, a mixture of acid salt and an aminoacid and a buffer, and a mixture of an alkali salt of an amino acid anda buffer. Additional buffering agents include citric acid, sodiumcitrate, sodium tartarate, sodium acetate, sodium carbonate, sodiumpolyphosphate, potassium polyphosphate, sodium pyrophosphate, potassiumpyrophosphate, disodium hydrogenphosphate, trisodium phosphate,tripotassium phosphate, sodium acetate, potassium metaphosphate,magnesium oxide, magnesium carbonate, magnesium silicate, calciumacetate, calcium glycerophosphate, calcium chloride, calcium hydroxide,calcium lactate, calcium carbonate, calcium bicarbonate, and othercalcium salts. Some buffering agents also impart effervescent qualitieswhen a powder is compounded into a liquid. In some embodiments, thevancomycin hydrochloride powder described herein, when compounded into aliquid form, comprises a buffering agent.

Preservatives include anti-microbials, anti-oxidants, and agents thatenhance sterility. Exemplary preservatives include ascorbic acid,ascorbyl palmitate, benzyl alcohol, BHA, BHT, citric acid, erythorbicacid, fumaric acid, malic acid, propyl gallate, sodium ascorbate, sodiumbenzoate, sodium bisulfate, sodium metabisulfite, sodium sulfite,parabens (methyl-, ethyl-, butyl-), benzoic acid, potassium sorbate, andvanillin. In some embodiments, the vancomycin hydrochloride powderdescribed herein, when compounded into a liquid form, comprises apreservative.

Sweeteners or sweetening agents include any compounds that provide asweet taste to make the product more palatable. This includes naturaland synthetic sugars, natural and artificial sweeteners (e.g.,sucralose), natural extracts and any material that initiates a sweetsensation in a subject. In some embodiments, the vancomycinhydrochloride powder described herein, when compounded into a liquidform, comprises a sweetener. In other embodiments, sweeteners in liquidform are used to solvate or dissolve the vancomycin hydrochloride powderdescribed herein.

Sugars illustratively include glucose, fructose, sucrose, xylitol,tagatose, maltitol, isomaltulose, lactitol, sorbitol, mannitol,erythritol, trehalose, maltodextrin, polydextrose, and the like. Othersweeteners include glycerin, inulin, maltol, acesulfame and saltsthereof, e.g., acesulfame potassium, alitame, aspartame, neotame, sodiumcyclamate, saccharin and salts thereof, e.g., saccharin sodium orsaccharin calcium, neohesperidin dihydrochalcone, stevioside, thaumatin,and the like. Sweeteners can be used in the form of crude or refinedproducts such as hydrogenated starch hydrosylates, maltitol syrup, highfructose corn syrup, and as branded proprietary blend products.Sweeteners can be used singly or combinations of two or more. Suitableconcentrations of different sweeteners can be selected based onpublished information, manufacturers’ data sheets, and by routinetesting. In certain instances, an above-described flavored solutioncomponent is used to solvate or dissolves the vancomycin hydrochloridepowder described herein.

In another embodiment, the liquid form comprises a flavoring agent orflavorant to enhance the taste or aroma of the solution component usedto solvate or dissolve the vancomycin hydrochloride powder describedherein. Suitable natural or synthetic flavoring agents can be selectedfrom standard reference books, such as Remington: The Science andPractice of Pharmacy (2000) and Fenaroli’s Handbook of FlavorIngredients (1994). Non-limiting examples of suitable natural flavors,some of which can be readily simulated with synthetic agents orcombinations thereof, include almond, anise, apple, apricot, banana,blackberry, blackcurrant, blueberry, caramel, cherry, chocolate,cinnamon, cranberry, grape, lemon, lime, orange, peppermint, pineapple,raspberry, spearmint, strawberry, vanilla, etc. Also useful,particularly where the composition is intended primarily for pediatricuse is tutti-frutti or bubble gum flavor, a compounded flavoring agentbased on fruit flavors. Presently, preferred flavoring agents includebubble gum, strawberry, cherry, grape, orange, peppermint, and vanilla.In some embodiments, the resultant liquid form from the vancomycinhydrochloride powder described herein comprises a grape (specifically,white grape) flavoring agent. According to pharmacist surveys that havebeen conducted, grape was among the most popular flavoring agents to beused for the compounding of oral vancomycin liquid (Vancomycin SurveyResults, 2013; Pharmacist Compounding Study, 2012). Flavoring agents maybe used singly or in combinations of two or more.

In further embodiments, the resultant liquid form from the vancomycinhydrochloride powder described herein comprises a coloring agent foridentity and/or aesthetic purposes. Suitable coloring agents approved bythe U.S. Food and Drug Administration (FDA) include FD&C Red No. 3, FD&CRed No. 20, FD&C Red No. 40, FD&C Yellow No. 6, FD&C Blue No. 2, D&CGreen No. 5, D&C Orange No. 5, D&C Yellow No. 10, caramel, ferric oxideand mixtures thereof. The resultant liquid form from the vancomycinhydrochloride powder described herein displays an amber-coloredappearance for identity and aesthetic purposes associated with a whitegrape flavor.

The vancomycin hydrochloride powder described herein is stable invarious storage conditions including refrigerated, ambient, andaccelerated conditions. Stable as used herein refer to vancomycinhydrochloride powder having microbial assay values of greater than 900or in some embodiments about 1100 µg/mg, compositions having about 85%vancomycin B and about 5% or less any other impurities or othersubstances and about 4.7% monodechlorovancomycin at the end of a givenstorage period. Stability may be assessed by cylinder-plate assay (thecylinder plate assay depends on the diffusion of the antibiotic from avertical cylinder through a solidified agar layer in a petri dish orplate. The growth of the specific microorganisms inoculated into theagar is prevented in a circular area or zone around the cylindercontaining the solution of the antibiotic), HPLC or any other knowntesting method. In some embodiments, the stable vancomycin hydrochloridepowder have microbial assay values of greater than 900, about 1100µg/mg, about 1050 µg/mg, or about 1000 µg/mg. In some embodiments, thestable vancomycin hydrochloride has composition of about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%vancomycin B. In some embodiments, the stable vancomycin hydrochloridepowder has about 5%, about 4%, about 3%, about 2.5%, about 2%, and 1.5%,about 1%, or about 0.5% individual impurities or substances. In yetother embodiments, the stable vancomycin hydrochloride powder has about4.7%, about 4.5%, about 4.0%, about 3.5%, about 3.0%, about 2.5%, about2.0%, about 1.5%, about 1.0%, or about 0.5% monodechlorovancomycin atthe end of a given storage period.

At ambient conditions, the vancomycin hydrochloride powder describedherein is stable, in some embodiments, for at least 1 month, at least 2months, at least 3 months, at least 6 months, at least 9 months, atleast 12 months, at least 15 months, at least 18 months, or at least 24months. In other embodiments, at accelerated conditions, the vancomycinhydrochloride powder described herein is stable for at least 1 month, atleast 2 months or at least 3 months. Accelerated conditions includetemperature and/or relative humidity (RH) that are above ambient levels(e.g. 25±5° C.; 55±10% RH). In some instances, an accelerated conditionis at about 30° C., about 35° C., about 40° C., about 45° C., about 50°C., about 55° C., or about 60° C. In other instances, an acceleratedcondition is above 65% RH, about 70% RH, about 75% RH, or about 80% RH.In further instances, an accelerated condition is about 40° C. or 60° C.at ambient humidity. In yet further instances, an accelerated conditionis about 40° C. at 75±5% RH humidity. Ambient conditions includetemperature and/or relative humidity (RH) that are at ambient levels(e.g. 25±5° C.; 55±10% RH. In some instances, an ambient condition is atabout 20° C., about 21° C., about 22° C., about 23° C., about 24° C.,about 25° C., about 26° C., about 27° C., about 28° C., about 29° C.,and about 30° C. In other instances, an ambient condition is about 45%RH, about 50% RH, about 55% RH, about 60% RH, or about 65% RH.Refrigerated conditions include temperature and/or relative humidity(RH) in typical refrigeration units (e.g. 5±3° C.). In some instances, arefrigerated condition is about 2° C., about 3° C., about 4° C., about5° C., about 6° C., about 7° C., or about 8° C.

The vancomycin hydrochloride powder described herein may be used for thepreparation or compounding of a vancomycin oral liquid. As provided inthe United States Pharmacopoeia (USP), oral liquids include, but are notlimited to, aqueous and nonaqueous solutions, suspensions, syrups,emulsions, elixirs, etc. It is envisioned that solutions are alsoincluded where certain components of the vancomycin oral liquidcompositions described herein are in solution or suspension. Toexemplify, when a vancomycin hydrochloride powder is compounded with theinactive ingredients of the aqueous solution component, the vancomycinhydrochloride powder particles disperse into the liquid form (e.g.,solution, suspension, etc.). Upon shaking the bottle containing thefinal product, the vancomycin hydrochloride powder particles aredispersed throughout the liquid form to provide a homogenous solutionfor consistent, accurate dosing for the patient. In some embodiments,the liquid form is a suspension. A thickening agent, or suspendingagent, may be added to prepare a suspension from a solution. Suspensionsconsist of flocculated particles, often containing the activeingredient, which are dispersed throughout the medium upon stirring,shaking, swirling, agitation, inversion, or a combination thereof.However, suspensions can cause the active ingredient to settle on thebottom of its container after standing for a period of time. Therefore,if not appropriately shaken, stirred, etc., inaccurate dosing for thepatient may occur. Furthermore, patients who require feeding tubes mayexperience clogging during administration which also significantlycontributes to rendering the pharmacotherapy ineffective. Solutions arehomogenous mixtures by which particles and/or molecules of the solute(s)(e.g., solids, liquids, or gases) are dissolved and uniformly dispersedthroughout the solvent. Though suspensions and solutions have their ownadvantages, the uniformity of a solution assures accurate dosage uponadministration; thus, this makes for a more preferred dosage form andwill less likely be problematic when administering through feedingtubes.

Liquid vehicles suitable for the vancomycin hydrochloride powderdescribed herein are selected for a particular oral liquid composition(e.g., solution, suspension, etc.) as well as other properties such asclarity, viscosity, compatibility with excipients, chemical inertness,palatability, odor, and color. Exemplary liquid vehicles include water,ethyl alcohol, glycerin, propylene glycol, syrup (e.g., sugar or othersweetener based, e.g., Ora-Sweet® SF sugar-free flavored syrup), juices(e.g., apple, orange, cranberry, cherry, tomato and the like), otherbeverages (e.g., tea, coffee, soft drinks, milk and the like), oils(e.g., olive, soybean, corn, mineral, castor and the like), andcombinations or mixtures thereof. Certain liquid vehicles, e.g., oil andwater, can be combined together to form emulsions. In some embodiments,water is used as a vehicle for a vancomycin oral liquid. In otherembodiments, syrup is used as a vehicle for a vancomycin oral liquid.For the vancomycin hydrochloride powder described herein, the solutioncomponent is used as the vehicle for a vancomycin oral liquid.

Mixing methods encompass any type of mixing that result in a homogenousvancomycin liquid composition. In some embodiments, a quantity of avancomycin hydrochloride powder is added to a liquid vehicle and thenmixed by a stirring, shaking, swirling, agitation, inversion, or acombination thereof. In certain instances, a liquid vehicle is added toa quantity of a vancomycin hydrochloride powder in a container (e.g., abottle, vial, bag, beaker, syringe, or the like). The container is thenmixed by stirring, shaking, swirling, agitation, inversion, or acombination thereof. In certain instances, a fractional volume of theliquid vehicle (e.g., one-half, one-third, one-fourth volume, etc.) isadded to a vancomycin hydrochloride powder, mixed by stirring, shaking,swirling, agitation, inversion, or a combination thereof; and, thesubsequent liquid fraction(s) is added and mixed. In certain instances,one-half fractional volume of liquid is added to a vancomycinhydrochloride powder in a container and mixing by shaking; the otherone-half fractional volume of the liquid vehicle is then subsequentlyadded and mixed. In any of the above embodiments, mixing (e.g.,stirring, shaking, swirling, agitation, inversion, or a combinationthereof) occurs for specific time intervals such as about 10 seconds,about 20 seconds, about 30 seconds, about 45 seconds, about 60 seconds,about 90 seconds, about 120 seconds, about 2.5 minutes, about 3 minutes,about 3.5 minutes, about 4 minutes, or about 5 minutes. In embodiments,where there are two or more mixing steps, the time intervals for eachmixing can be the same (e.g., 2×60 seconds) or different (e.g., 60seconds for the first mixing and 30 seconds for the second mixing).

The vancomycin oral liquid compositions described herein are stable invarious storage conditions including refrigerated and ambientconditions. Stable compositions as used herein refer to vancomycin oralliquid compositions having at least 98% vancomycin at the end of a givenstorage period. In some embodiments stable compositions have at least85%, 90%, 93%, 95%, 96%, 97%, or 99% vancomycin at the end of a givenstorage period. Stability may be assessed by functional microbial assaysor any other known testing method. In some embodiments, the stablevancomycin oral liquid compositions have at least about 98%, at leastabout 99%, at least about 100%, at least about 101%, at least about102%, at least about 103%, at least about 104%, at least about 105%, atleast about 106%, at least about 107%, at least about 108%, at leastabout 109%, at least about 110%, at least about 111%, at least about112%, at least about 113%, at least about 114%, at least about 115%Vancomycin at the end of a given storage period using a functionalmicrobial assay. Since the assay assesses actual versus theoreticalmicrobial count, the percent of active vancomycin measured in thesolution may exceed 100%. As shown in Example 4, the results indicatethat the vancomycin oral liquid composition when stored at refrigerated(2 - 8° C.) conditions is stable for at least 30 days. In someembodiments, the stable vancomycin oral liquid composition have at leastabout 96.9% vancomycin at the end of a given storage period using achemical assay.

At refrigerated and ambient conditions, in some embodiments, thevancomycin oral liquid compounding compositions described herein arestable for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or atleast 6 weeks. Ambient conditions include temperature and/or relativehumidity (RH) that are at ambient levels (e.g. 25±5° C.; 55±10% RH). Insome instances, an ambient condition is at about 20° C., about 21° C.,about 22° C., about 23° C., about 24° C., about 25° C., about 26° C.,about 27° C., about 28° C., about 29° C., and about 30° C. In otherinstances, an ambient condition is about 45% RH, about 50% RH, about 55%RH, about 60% RH, or about 65% RH. Refrigerated conditions includetemperature and/or relative humidity (RH) in typical refrigeration units(e.g. 5±3° C.). In some instances, a refrigerated condition is about 2°C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., orabout 8° C. In other embodiments the liquid solutions are stable atambient and refrigerated conditions for several months.

For the vancomycin hydrochloride powder and liquid compositionsdescribed herein, kits and articles of manufacture are also described.

Kits may include vancomycin hydrochloride powder equivalent to 1 mg to100 mg/mL vancomycin, more preferably 5 mg to 20 mg/mL vancomycin, morepreferably 10 mg to 75 mg/mL, more preferably 20 mg to 60 mg/mL, andmost preferably 25 mg/mL and 50 mg/mL.

Kits may be packaged to prepare volumes appropriate for requiredtherapy, also referred to as unit of use containers. Exemplary volumesinclude 1 ounce (30 mL) to 20 ounces (600 mL), more preferably 3 ounces(60 mL) to 15 ounces (450 mL), and most preferably 5 ounces (150 mL), 7ounces (210 mL), and 10 ounces (300 mL). The typical treatment for C.difficile pseudomembranous colitis and/or Staphylococcal enterocolitiscan be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 14 days, and/or longer forrecurrent infections, according to a survey study among pharmacists invarious practice settings (Vancomycin Volume Study, April 2013).

An exemplary kit is shown in FIG. 1 . Such kits can include a firstcontainer (1), a second container (2), a packaging material (3), andinstructions for use (4) either included within the package or on thepackage. The package may be compartmentalized to receive one or morebottles and the like, each of the container(s) comprising of one of theseparate elements to be used in a method described herein including avancomycin hydrochloride powder or solution component. The suitablecontainers can be formed from a variety of materials including plasticmaterials.

A kit will typically be comprised of one or more additional containers,each one with one or more of various materials (such as vancomycinhydrochloride powder and solution component containing inactiveingredients) desirable from a commercial and user standpoint for avancomycin hydrochloride powder or solution component herein.Non-limiting examples of such materials include, but not limited to,buffers, diluents, tools; carrier, package, container, and/or bottlelabels listing contents, and package inserts with a set of instructionsfor compounding are included.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers, or characters forming the label areattached or stamped onto the container itself; a label can be associatedwith a container when it is present within a receptacle or carrier thatalso holds the container, e.g., as a package insert.

The vancomycin oral liquid compositions may be used for the treatment ofdiseases and conditions described herein. In addition, a method fortreating any of the diseases or conditions described herein in a subjectin need of such treatment involves administration of vancomycin oralliquid compositions in therapeutically effective amounts to the subject.In some embodiments, the amount of a given vancomycin oral liquidcomposition that corresponds to such an amount varies depending onfactors such as the particular vancomycin salt or form, diseasecondition and its severity, the identity (age, weight, sex) of thesubject or patient in need of treatment, but can nevertheless bedetermined according to the particular circumstances surrounding thecase, including, e.g., the specific agent being administered, the liquidcomposition type, the condition being treated, and the subject orpatient being treated.

In further embodiments, the daily dosages appropriate for the vancomycinoral liquid compositions described herein are from about 40 mg/kg perbody weight. In one embodiment, the daily dosage appropriate for thevancomycin liquid compositions is about 500 mg to 2 g.

In some embodiments, the vancomycin oral liquid compositions describedherein are for the treatment of severe and/or recurrent infections ofClostridium difficile pseudomembranous colitis.

The treatment of certain diseases or conditions (e.g., C. difficilepseudomembranous colitis, Staphylococcal enterocolitis, etc.) in apatient or subject with a vancomycin oral liquid composition describedherein encompass additional therapies and treatment agents in someembodiments. Such additional therapies and treatment regimens includeanother therapy, e.g., additional antibiotics, for the treatment of theparticular disease in some embodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice of testing ofembodiments described herein, certain preferred methods, devices, andmaterials are now described.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “an excipient” is a referenceto one or more excipients and equivalents thereof known to those skilledin the art, and so forth.

The term “about” is used to indicate that a value includes the standardlevel of error for the device or method being employed to determine thevalue. The use of the term “or” in the claims is used to mean “and/or”unless explicitly indicated to refer to alternatives only or thealternatives are mutually exclusive, although the disclosure supports adefinition that refers to only alternatives and to “and/or”. The terms“comprise”, “have”, and “include” are open-ended linking verbs. Anyforms or tenses of one or more of these verbs “comprises,” “comprising,”“has,” “having,” “includes,” and “including” are also open-ended. Forexample, any method that “comprises,” “has” or “includes” one or moresteps is not limited to possessing only those one or more steps and alsocovers other unlisted steps.

“Optional” or “optionally” may be taken to mean that the subsequentlydescribed structure, event or circumstance may or may not occur, andthat the description includes instances where the events occurs andinstances where it does not.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. In some embodiments, a therapeutic agent such as oralvancomycin is directed to the treatment and/or the amelioration of,reversal of, or stabilization of the symptoms of Clostridium difficilepseudomembranous colitis and/or Staphylococcal enterocolitis describedherein.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic systemically or locally, as directly into oronto a target tissue, or to administer a therapeutic to a patientwhereby the therapeutic positively impacts the tissue to which it istargeted. Thus, as used herein, the term “administering”, when used inconjunction with an oral vancomycin composition, can include, but is notlimited to, providing an oral vancomycin composition into or onto thetarget tissue; providing an oral vancomycin composition systemically toa patient by, e.g., oral administration whereby the therapeutic reachesthe target tissue or cells. “Administering” a composition may beaccomplished by injection, topical administration, and oraladministration or by other methods alone or in combination with otherknown techniques.

As used herein, the term “container” or “containers” may mean bottles,vials, jars, pouches, and the like.

As used herein, the terms “patient,” “subject” and “individual” areintended to include living organisms in which certain conditions asdescribed herein can occur. Examples include humans, monkeys, cows,sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. Ina preferred embodiment, the patient is a primate. In certainembodiments, the primate or subject is a human. In certain instances,the human is an adult. In certain instances, the human is child. Incertain instances, the human is elderly. In other instances, the humanis 65 years of age or older. Other examples of subjects includeexperimental animals such as mice, rats, dogs, cats, goats, sheep, pigs,and cows. The experimental animal can be an animal model for a disorder,e.g., a transgenic mouse with Clostridium difficile pseudomembranouscolitis and Staphylococcal enterocolitis pathology. A patient can be ahuman suffering from Clostridium difficile pseudomembranous colitis,Staphylococcal enterocolitis, or its variants or etiological forms.

By “pharmaceutically acceptable”, it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The term “pharmaceutical composition” shall mean a compositioncomprising of at least one active ingredient, whereby the composition isamenable to investigation for a specified, efficacious outcome in amammal (for example, without limitation, a human). Those of ordinaryskill in the art will understand and appreciate the techniquesappropriate for determining whether an active ingredient has a desiredefficacious outcome based upon the needs of the artisan.

A “therapeutically effective amount” or “effective amount” as usedherein refers to the amount of active compound or pharmaceutical agentthat elicits a biological or medicinal response in a tissue, system,animal, individual or human that is being sought by a researcher,veterinarian, medical doctor or other clinician, which includes one ormore of the following: (1) preventing the disease; for example,preventing a disease, condition or disorder in an individual that may bepredisposed to the disease, condition or disorder but does not yetexperience or display the pathology or symptomatology of the disease,(2) inhibiting the disease; for example, inhibiting a disease, conditionor disorder in an individual that is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,arresting further development of the pathology and/or symptomatology),and (3) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual that is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., reversing the pathology and/or symptomatology). As such,a non-limiting example of a “therapeutically effective amount” or“effective amount” of a composition of the present disclosure may beused to inhibit, block, or reverse the activation, migration, orproliferation of cells or to effectively treat Clostridium difficilepseudomembranous colitis and/or Staphylococcal enterocolitis orameliorate the symptoms of Clostridium difficile pseudomembranouscolitis and/or Staphylococcal enterocolitis.

The terms “treat,” “treated,” “treatment,” or “treating” as used hereinrefers to both therapeutic treatment in some embodiments andprophylactic or preventative measures in other embodiments, wherein theobject is to prevent or slow (lessen) an undesired physiologicalcondition, disorder or disease, or to obtain beneficial or desiredclinical results. For the purposes described herein, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms; diminishment of the extent of the condition, disorder ordisease; stabilization (i.e., not worsening) of the state of thecondition, disorder or disease; delay in onset or slowing of theprogression of the condition, disorder or disease; amelioration of thecondition, disorder or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder or disease. Treatment includeseliciting a clinically significant response without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment. A prophylactic benefit oftreatment includes prevention of a condition, retarding the progress ofa condition, stabilization of a condition, or decreasing the likelihoodof occurrence of a condition. As used herein, “treat,” “treated,”“treatment,” or “treating” includes prophylaxis in some embodiments.

Embodiments have been described where the techniques are implemented incircuitry and/or computer-executable instructions. It should beappreciated that some embodiments may be in the form of a method, ofwhich at least one example has been provided. The acts performed as partof the method may be ordered in any suitable way. Accordingly,embodiments may be constructed in which acts are performed in an orderdifferent than illustrated, which may include performing some actssimultaneously, even though shown as sequential acts in illustrativeembodiments.

Various aspects of the embodiments described above may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1: Stability of Vancomycin Hydrochloride Powder

The stability of vancomycin hydrochloride powder was evaluated. 10 g ofpowder was placed in 325 mL HDPE (high density poly ethylene) bottle andstored under 25° C. /60% Relative Humidity (RH) and 40° C. /75% RH. Atvarious time points the powder in the bottle was analyzed for vancomycinhydrochloride, vancomycin B, other impurities, andmonodechlorovancomycin by HPLC or cylinder plate analysis. The followingtables depict the stability of vancomycin hydrochloride in the bottle atthe various storage conditions.

µg/mg vancomycin hydrochloride powder Time (Months) 40° C. /75% RH 01067 mcg/mg 1 1076 mcg/mg 2 1097 mcg/mg 3 1094 mcg/mg 6 1137 mcg/mg

% Vancomycin B powder in bottle Time (Months) 25° C. /60% (RH) 40° C./75% RH 0 93.03% 93.03% 1 --- 91.87% 2 --- 89.14% 3 92.19% 88.22% 692.04% 81.84% 9 89.34% --- 12 88.85% --- 18 90.8% ---

% other impurities powder in bottle Time (Months) 25° C. /60% (RH) 40°C. /75% RH 0 <5.0% for all other <5.0% for all other 1 --- <5.0% for allother 2 --- <5.0% for all other 3 <5.0% for all other <5.0% for allother 6 <5.0% for all other <5.0% for all other 9 <5.0% for all other--- 12 <5.0% for all other --- 18 <5.0% for all other ---

% Monodechlorovancomycin powder in bottle Time (Months) 40° C. /75% RH 11.0% 2 1.3% 3 1.6%

Based on the powder in bottle stability at ambient and acceleratedconditions (25° C. /60%RH and 40°/75%RH), it was determined that thevancomycin hydrochloride powder was stable.

Example 2: Stability of Prepared Vancomycin Oral Liquid

50 mg/mL vancomycin oral liquids were prepared from vancomycinhydrochloride made with the addition of the solution component andplaced under refrigerated and 25° C./60%RH-ambient conditions. Thefollowing tables depict the stability of vancomycin oral liquid atvarious storage conditions.

% vancomycin Vancomycin Oral Liquid Time (Days) Refrigerated 25° C. /60%(RH) 0 109.7% 109.7% 14 109.2% 109.8% 30 110.0% 109.4%

In the prepared liquids, the vancomycin oral liquid was most stable atrefrigerated and 25° C. /60%RH conditions.

Example 3: Reconstitution Method

The overall mixing efficiency and solution drug concentration wasevaluated. 289 mL of the solution component was added to a 325 mL bottlecontaining 15.38 g of vancomycin hydrochloride powder and the bottle wasvigorously shaken for 30 seconds.

The resultant liquid was a clear amber solution. (The amber color is dueto a color additive to the solution component.) The vancomycinhydrochloride powder easily dissolves in the solution component.

Example 4: Additional Stability Studies

Vancomycin hydrochloride was further investigated in stability studies.15.38 g of vancomycin hydrochloride powder in 325 mL white plasticbottled were examined for stability in 25±2° C. /60±5% RH for up to 3months and 40±2° C. /75±5% RH (accelerated) conditions up to 3 months.At each given time point, vancomycin hydrochloride, vancomycin B, otherimpurities, and monodechlorovancomycin were assayed by HPLC or totalmicrobiological activity was assessed by cylinder plate analysis. Thebelow tables depict stability of the powder at the various conditions.

Vancomycin Hydrochloride Powder-25° C./60%RH Time (Months) VancomycinVancomycin B Other Impurities Monodechlororvancomycin 0 1,112 mcg/mg94.7% <5.0% for all other 1.5% 3 1,070 mcg/mg 91.8% <5.0% for all other1.1%

Vancomycin Hydrochloride Powder-40° C./60%RH Time (Months) VancomycinVancomycin B Other Impurities Monodechlororvancomycin 0 1,112 mcg/mg94.7% <5.0% for all other 1.5% 1 1,095 mcg/mg 91.3% <5.0% for all other1.4% 2 1,108 mcg/mg 90.8% <5.0% for all other 1.1% 3 1,095 mcg/mg 87.6%<5.0% for all other 1.0%

Vancomycin hydrochloride powder remained stable through 3 months underaccelerated stability conditions and 3 months under ambient (25±2° C./60±5% RH) with slight decrease in the levels of vancomycin B. However,none of the other impurities exceeded 5.0% nor did theMonodechlorovancomycin exceed 4.7%. Stability at ambient conditions wasalso examined for three additional lots of vancomycin hydrochloridepowder at lower concentrations/smaller bottle sizes. Similar resultswere observed for the levels of vancomycin B.

Reconstituted Liquid Stability: Vancomycin hydrochloride powder wasreconstituted in 325 mL bottles according to mixing method (30 secondsof shaking) and stored at refrigerated and ambient 25±2° C. /60±5% RHconditions. Aliquots were taken during selected time points during thestudy period. The below tables depict stability of the reconstitutedliquid at various conditions.

% vancomycin Vancomycin Oral Liquid Time (Days) Refrigerated 25° C. /60%(RH) 0 103.2% 102.9% 7 103.7% 102.9% 14 104.3% 104.2% 21 107.2% 106.4%30 108.6% 106.1%

pH Vancomycin Oral Liquid Time (Days) Refrigerated 25° C. /60% (RH) 03.0 3.0 7 2.8 2.8 14 2.9 3.0 21 3.1 3.2 30 3.0 3.2

After being reconstituted, the vancomycin oral liquid was stable atrefrigerated and ambient conditions with essentially unchanged values ofits attributes up to the end of the study period (30 days).

The solution component is tested for description, pH, density, sodiumbenzoate assay, container/closure, and microbial limits. This testingwas also performed at specified stability intervals. All testingreflects unchanged values of attributes up to the end of shelf life.

Example 5: Antimicrobial Effectiveness Testing Stability

Vancomycin hydrochloride powder was reconstituted in 325 mL bottlesaccording to mixing method (30 seconds of shaking) and stored atrefrigerated and ambient 25±2° C. /60±5% RH conditions. Aliquots weretaken during selected time points during the study period. The belowtables depicts the antimicrobial effectiveness testing.

Vancomycin Oral Liquid Time 0 25° C. /60% (RH) Test Organism InitialInoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml 28 day %red^(∗) 28 day E.coli 4.9×10⁵ <10 >99.99 <10 >99.99 C. albicans 4.1×10⁵ <10 >99.99<10 >99.99 A. brasiliensis 2.6×10⁵ <10 >99.99 <10 >99.99 Time 7 day 25°C. /60% (RH) Test Organism Initial Inoculum CFU/ml 14 day %red^(∗) 14day CFU/ml 28 day %red^(∗) 28 day E. coli 5.8×10⁵ <10 >99.99 <10 >99.99C. albicans 3.9×10⁵ <10 >99.99 <10 >99.99 A. brasiliensis 3.5×10⁵<10 >99.99 <10 >99.99 Time 14 day 25° C. /60% (RH) Test Organism InitialInoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml 28 day %red^(∗)* 28 day E.coli 4.5×10⁵ <10 >99.99 <10 >99.99 C. albicans 2.9×10⁵ <10 >99.99<10 >99.99 A. brasiliensis 3.8×10⁵ <10 >99.99 <10 >99.99 Time 30 day 25°C. /60% (RH) Test Organism Initial Inoculum CFU/ml 14 day %red^(∗) 14day CFU/ml 28 day %red^(∗) 28 day E. coli 4.6×10⁵ <10 >99.99 <10 >99.99C. albicans 3.6×10⁵ <10 >99.99 <10 >99.99 A. brasiliensis 3.9×10⁵<10 >99.99 <10 >99.99 ^(∗)% reduction Vancomycin Oral Liquid Time 0 25°C. /60% (RH) Test Organism Initial Inoculum CFU/ml 14 day %red^(∗) 14day CFU/ml 28 day %red^(∗) 28 day E. coli 4.9×10⁵ <10 >99.99 <10 >99.99C. albicans 4.1×10⁵ <10 >99.99 <10 >99.99 A. brasiliensis 2.6×10⁵1.2×10³ 99.54 1.3×10² 99.95 Time 7 day 25° C. /60% Test Organism InitialInoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml 28 day %red^(∗) 28 day E.coli 5.8×10⁵ <10 >99.99 <10 >99.99 C. albicans 3.9×10⁵ <10 >99.99<10 >99.99 A. brasiliensis 3.5×10⁵ 2.3×10³ 99.34 3.1×10² 99.91 Time 14day 25° C. /60% Test Organism Initial Inoculum CFU/ml 14 day %red^(∗) 14day CFU/ml 28 day %red^(∗) 28 day E. coli 4.5×10⁵ <10 >99.99 <10 >99.99C. albicans 2.9×10⁵ <10 >99.99 <10 >99.99 A. brasiliensis 3.8×10⁵2.9×10³ 99.24 5.0×10¹ 99.99 Time 30 day 25° C. /60% Test OrganismInitial Inoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml 28 day %red^(∗) 28day E. coli 4.6×10⁵ <10 >99.99 <10 >99.99 C. albicans 3.6×10⁵1.0×10¹ >99.99 <10 >99.99 A. brasiliensis 3.9×10⁵ 1.1×10³ 99.72<10 >99.99 ^(∗)% reduction

Example 6: Antimicrobial Effectiveness Testing Stability forExtemporaneous Compounding

The most common extemporaneous compounding method for vancomycin oralliquid is to reconstitute lyophilized vancomycin HCL powder forinjection with sterile water. Lyophilized vancomycin hydrochloridepowder for injection was reconstituted in 325 mL bottles and stored atrefrigerated and ambient 25±2° C. /60±5% RH conditions. Aliquots weretaken during selected time points during the study period. The belowtables depicts the antimicrobial effectiveness testing. The testrequirement is to assure no growth in either yeast or mold is observedthroughout the inoculation. The extemporaneous compounding method failsto meet these criteria. If contamination were to occur theextemporaneous compounding method does not preserve against mold oryeast.

Vancomycin Oral Liquid Time 0 25° C. /60% (RH) Test Organism InitialInoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml 28 day %red^(∗) 28 day E.coli 4.9×10⁵ <10 >99.99 <10 >99.99 C. albicans 4.1×10⁵ 1.9×10³ 99.541.8×10³ 99.56 A. brasiliensis 2.6×10⁵ 2.8×10⁵ 0** 8.0×10⁴ 69.23 Time 7day 25° C. /60% (RH) Test Organism Initial Inoculum CFU/ml 14 day%red^(∗) 14 day CFU/ml 28 day %red^(∗) 28 day E. coli 5.8×10⁵ <10 >99.99<10 >99.99 C. albicans 3.9×10⁵ 5.2×10³ 98.67 1.65×10³ 99.58 A.brasiliensis 3.5×10⁵ 3.6×10⁵ 0** 3.2×10⁵ 8.57 Time 14 day 25° C. /60%(RH) Test Organism Initial Inoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml28 day %red^(∗) 28 day E. coli 4.5×10⁵ <10 >99.99 <10 >99.99 C. albicans2.9×10⁵ 4.1×10³ 98.59 4.0×10³ 98.62 A. brasiliensis 3.8×10⁵ 2.7×10⁵28.95 2.5×10⁵ 34.21 Time 30 day 25° C. /60% (RH) Test Organism InitialInoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml 28 day %red^(∗) 28 day E.coli 4.6×10⁵ <10 >99.99 <10 >99.99 C. albicans 3.6×10⁵ 8.2×10⁴ 77.223.5×10⁴ 90.28 A. brasiliensis 3.9×10⁵ 2.9×10⁵ 25.64 1.8×10⁵ 53.85Vancomycin Oral Liquid Time 0 Refrigerated Test Organism InitialInoculum CFU/ml 14 day %red^(∗) 14 day CFU/ml 28 day %red^(∗) 28 day E.coli 4.9×10⁵ <10 >99.99 <10 >99.99 C. albicans 4.1×10⁵ 9.7×10³ 97.638.6×10³ 97.90 A. brasiliensis 2.6×10⁵ 3.2×10⁵ 0** 1.6×10⁵ 38.46 Time 7day Refrigerated Test Organism Initial Inoculum CFU/ml 14 day %red^(∗)14 day CFU/ml 28 day %red^(∗) 28 day E. coli 5.8×10⁵ <10 >99.99<10 >99.99 C. albicans 3.9×10⁵ 5.5×10⁴ 83.33 8.2×10⁴ 78.97 A.brasiliensis 3.5×10⁵ 3.8×10⁵ 0** 3.0×10⁵ 14.29 Time 14 day RefrigeratedTest Organism Initial Inoculum CFU/ml14 day %red^(∗)14 day CFU/ml28 day%red^(∗)28 day E. coli 4.5×10⁵ <10 >99.99 <10 >99.99 C. albicans 2.9×10⁵1.5×10⁴ 94.79 <10 >99.99 A. brasiliensis 3.8×10⁵ 2.6×10⁵ 31.58 2.0×10⁵47.37 Time 30 day Refrigerated Test Organism Initial Inoculum CFU/ml14day %red^(∗)14 day CFU/ml28 day %red^(∗)28 day E. coli 4.6×10⁵<10 >99.99 <10 >99.99 C. albicans 3.6×10⁵ 1.3×10⁴ 96.39 1.0×10² 99.97 A.brasiliensis 3.9×10⁵ 2.6×10⁵ 33.33 1.7×10⁵ 56.41 ^(∗)% reduction^(∗∗)Fails criteria Category 3 (yeast and mold): no increase from theinitial calculated count at 14 and 28 days.

Example 7: Long Term Stability Analysis

The stability of the vancomycin hydrochloride solution of the inventionwas compared directly to the stability of a vancomycin hydrocholoridesolution made from a sterile lyophilized powder typically used formaking an injectable solution. The results are shown in the followingTable.

Solution Using Sterile Lyophilized Powder Solution of the InventionVancomyn HCl 145 mL of sterile water for injection Vancomyn HCl 145 mLliquid formulation (50 mg/mL Solution) (50 mg/mL Solution) AppearanceAppearance pH Precipitate Amber pH Precipitate Days Light tan color Time0 3.23 1 1 Amber 3.2 1 2 Light tan color Amber 3 Light tan color 3.2 1 43.2 1 5 6 Light tan color 7 Light tan color 3.2 1 3 1 8 3.2 1 3.1 1 9Amber 10 Light tan color 11 3.2 1 3 1 12 Amber 13 Light tan color Amber14 Light tan color 3.2 1 Amber 3 1 15 Light tan color 3.2 1 Amber 3.2 116 Light tan color 3.2 1 Amber 3.1 1 17 Light tan color 3.2 1 3.1 1 183.2 1 3.1 1 19 Amber 20 Light tan color Amber 21 Light tan color 3.2 1Amber 3.1 1 22 Light tan color 3.2 1 Amber 3.2 1 23 Light tan color 3.21 Amber 3.2 1 24 Light tan color 3.2 1 3.1 1 25 3.2 1 3.2 1 26 Amber 27Light tan color Amber 28 Light tan color 3.2 1 Amber 3.1 1 29 Light tancolor 3.2 1 Amber 3.1 1 30 Light tan color 3.2 1 Amber 3.1 1 31 Lighttan color 3.2 1 3.2 1 32 3.2 1 3.2 1 33 Amber 34 Light tan color Amber35 Light tan color 3.2 1 Amber 3.1 1 36 Light tan color 3.2 1 Amber 3.11 37 Light tan color 3.2 1 Amber 3.1 1 38 Light tan color 3.2 1 3.2 1 393.2 1 Amber 3.2 1 40 Amber 41 Light tan color 3.2 1 42 Light tan color3.2 1 3.2 1 43 Out 3.2 2 Amber 44 Light tan color Amber 45 Light tancolor 3.2 2 3.2 1 46 3.2 3 3.2 1 47 Amber 48 Light tan color Amber 49Taken for analysis 3.2 4 Amber 3.2 1 50 3.2 4 Amber 3.2 1 51 3.2 1 523.2 1 53 3.2 1 54 55 Holiday 56 Out Amber 57 Amber 58 Amber 3.2 2 59 3.23 60 3.2 4 1 = Free of particles 2 = Small amount of particles visible 3= Large amount of particles visible 4 = Completely out of solution

At day 43 for the vancomycin solution with lyophilized powder smallamounts of particles were visible. At day 49 the particles were abundantand were observed to be completely out of solution.

In contrast, the solution of the invention was particle free through atleast day 58, when a small amount of particles were visible. It remainedin solution through day 60.

Example 8: Long Term Stability Analysis of Three Different Preparationsof Vancomycin Stored at Ambient and Refrigerated Temperatures for up to30 days

The stability of the vancomycin hydrochloride solution of the inventionwas compared directly to the stability of a vancomycin hydrocholoridesolution made from a sterile lyophilized powder typically used formaking an injectable solution and vancomycin capsules ground to a powderand reconstituted. To determine the impact of storage on the potency ofvancomycin, the different formulations were tested at the time ofpreparation, after 14 and 30 days, stored at ambient and refrigeratortemperatures by measuring MICs for 25 strains of C. difficile. Thecontrol was vancomycin reference standard powder that had beenreconstituted to prepare a stock solution, then stored at -70 C. Thevancomycin hydrochloride solution of the invention and the IV solutionwere reconstituted to a concentration of 25 mg/ml. The generic“capsules” were tablets that had to be ground to a fine powder beforethey would dissolve in water. The MICs were determined by the referenceagar dilution method (CLSI M11-A8). All serial two-fold dilutions wereprepared on the same day and added to molten agar to prepare plates.

The following materials were used in the study:

-   Vancomycin from Cutis Pharma Inc. Lot E836 reconstituted to 25 mg/ml-   Vancomycin for IV reconstituted to 25 mg/ml Hospira Inc. Lot    322578EO4-   Vancomycin capsules Prasco Lot 453019, ground to a powder and    reconstituted to 25 mg/ml-   Vancomycin laboratory standard powder, Sigma, Lot SLBD44270-   Brucella deeps, Anaerobe Systems (Morgan Hill, CA) Lot 3551809-   Hemostat Sheep blood Lot 199032-15-   Brucella blood agar plates, Hardy Diagnostics (Santa Maria, CA) Lot    14064

The in vitro activity was tested for three different preparations ofvancomycin freshly reconstituted, and stored at ambient and atrefrigerated temperatures. The C. difficile strains were selected torepresent the most frequently encountered ribotypes. MICs for thepreparations were measured by agar dilution according to the proceduresin the CSI M11-A8 document (Clinical and Laboratory Standards Institute.2012. Methods for antimicrobial susceptibility testing of anaerobicbacteria; approved standard-eighth edition. CLSI document M11-A8. CLSI,Wayne, Pa). Quality control strains included Clostridium difficile ATCC700057 and Staphylococcus aureus ATCC 29213.

The organisms were recovered from clinical samples, identified bystandard methods (Jousimies-Somer, H. R., et al 2002. Wadsworth-KTLAnaerobic Bacteriology Manual. Star Publishing, Belmont, CA.) and storedin 20% skim milk at -70° C. They were taken from the freezer andsubcultured at least twice on supplemented Brucella agar and incubatedunder anaerobic conditions at 37° C. for 24 h prior to testing. Inoculawere prepared by direct suspensions of colonies into Brucella broth toequal the turbidity of the 0.5 McFarland standard.

Plates were prepared by adding 1 ml of lysed sheep blood and two mls ofthe twofold dilutions of the vancomycin preparations to molten agar.Plates were mixed, poured and allowed to harden. Suspensions of theorganisms were applied to the plates using a Steers replicating devicethat delivered approximately 10⁵ cfu/spot. Each preparation was testedin triplicate. Plates were incubated at 37° C. for 48 h.

Plates were examined for growth. The MIC was defined as theconcentration that completely inhibited growth or caused a majorreduction in the appearance of the button compared to the drug-freecontrol plate.

The results are shown in the following Tables. Table 1 presents theresults as the most frequent of the triplicates. Table 2 presents theaverage of the three MICs.

The triplicate MICs (µg/ml) were averaged for each organism and thegeometric mean was calculated for all 25 strains, preparations and timeperiods. The MICs for each preparation at T(0), 14 d and 30 d atambient/refrigerator were as follows: vancomycin solution of theinvention 1.9, 1.5/1.4, 2.2/1.8; IV solution 2.2,1.5/1.5, 2.2/2.1;crushed tablets 2.2, 1.5/1.5, 2.3/2.0, respectively. The referencemethod was 1.5 µg/ml. The differences were less than one dilution forall of the values and were considered insignificant. MICs showed minimalvariation among all preparations over the study period. However, theaverage MICs of the vancomycin solution of the invention were shown tobe lower than the other preparations in several instances, as shown inTable 3 below and FIG. 2 . FIG. 2 is a Bar graph showing the average MICvalues for each of the solutions at each of the time points. Vancomycinwas remarkably stable in the preparations, regardless of storageconditions, for up to 30 days.

TABLE 1 C. difficile vancomycin MICs from triplicate test. Results shownas the best of 3 values Solution of the Invention IV RMA# RIBOTYPE Fresh14 d 30 d Fresh 14 d 30 d RT REFRIG RT REFRIG RT REFRIG RT REFRIG 24608002 2 1 1 2 2 2 1 1 2 2 24605 002 2 1 1 2 2 2 1 1 2 2 24655 005 2 1 1 22 2 2 2 2 2 24615 005 2 2 2 2 2 2 2 2 4 2 24678 012 4 4 4 4 4 4 4 4 4 424628 012 2 1 1 2 2 2 1 1 2 2 24527 014 1 1 1 2 1 2 1 1 2 2 24529 014 11 1 2 1 2 1 1 2 1 24547 014 2 1 1 2 2 2 1 1 2 2 24541 020 1 1 1 2 1 2 11 2 2 24543 020 1 1 1 2 1 2 1 1 2 2 24567 020 2 1 1 2 2 2 1 2 2 2 24504027 2 1 1 2 1 2 1 1 2 2 24510 027 2 1 1 2 1 2 1 1 2 2 24519 027 1 1 1 21 1 1 1 2 2 24683 027 2 2 2 2 1 2 2 2 2 2 24690 027 4 4 4 4 4 4 4 4 4 424561 078 1 1 1 1 1 1 1 1 1 1 24575 078 1 1 1 2 1 1 1 1 2 1 24601 106 21 1 2 2 2 2 1 2 2 24607 106 2 1 1 2 2 2 1 1 2 2 24594 106 4 4 4 4 4 4 44 4 4 24641 001/072 2 1 1 2 2 2 1 1 2 2 24643 001/072 2 1 1 2 2 2 1 1 22 24646 001/072 2 1 1 2 2 2 1 1 2 2 ATCC 7000057 QC 2 1 1 2 2 2 2 2 2 2ATCC 29213 SA QC 2 1 2 2 RMA# Capsules Fresh 14D RT 14D REFRIG 30D RT30D REFRIG Reference Standard Fresh 24608 002 2 1 1 2 2 1 24605 002 2 11 2 2 1 24655 005 2 2 1 2 2 2 24615 005 2 2 2 2 2 2 24678 012 4 4 4 4 44 24628 012 2 1 1 2 2 1 24527 014 2 1 1 2 1 1 24529 014 2 1 1 2 1 124547 014 2 1 1 2 1 1 24541 014 2 1 1 2 2 1 24543 020 2 1 1 2 2 1 24567020 2 1 1 2 2 1 24504 027 2 1 1 2 2 1 24510 027 2 1 1 2 2 1 24519 027 21 1 2 1 1 24683 027 2 2 2 2 2 2 24690 027 4 4 4 4 4 4 24561 078 1 1 1 11 1 24575 078 1 1 1 2 1 1 24601 106 2 2 1 2 2 2 24607 106 2 1 1 2 2 124594 106 4 4 4 4 4 4 24641 001/072 2 1 1 2 2 1 24643 001/072 2 1 1 2 21 24646 001/072 2 1 1 2 2 1 ATCC 7000057 QC 2 2 1 2 2 1 C. difficileATCC 29213 SA CQ 2 1 2 S. aureus

TABLE 2 C. difficile Vancomycin susceptibilities – average of triplicateMIC values Solution of the Invention IV RMA# RIB OTY PE Fresh 14d 30dFresh 14d 30d RT REFRI G RT REFRI G RT REFRIG RT REFRIG 24608 002 2 1 12 2 2 1 1 2 2 24605 002 2 1.1 1 2 2 2 1 1 2 2 24655 005 1.7 3 1.3 2 2 21.7 2 2 2 24615 005 2 2 1.7 2 2 2.7 2 2 3.3 2 24678 012 4 4 4 4 4 4 4 44 4 24628 012 2 1 1 2 2 2.7 1 1 2 2 24527 014 1 1 1 2 1 1.7 1 1 2 1.724529 014 1 1 1 2 1 1.7 1 1 2 1.3 24547 014 2 1 1 2 2 2 1 1 2 2 24541020 1.3 1 1 2 1 2 1 1 2 1.7 24543 020 1 1 1 2 1 2.7 1 1 2 1.7 24567 0202 1 1 2 2 2 1 1.7 2 2 24504 027 1.7 1 1 2 1 2 1 1 2 2 24510 027 1.7 1 12 1 2 1 1 2 2 24519 027 1 1 1 2 1 1.3 1 1 2 1.7 24683 027 1.7 2 2 2 1 22 2 2 2 24690 027 4 4 4 4 4 4 4 4 4 4 24561 078 1 1 1 1 1 1 1 1 1 124575 078 1 1 1 2 1 1.3 1 1 1.7 1 24601 106 2 1 1 2 2 2 1.7 1 2 2 24607106 2 1 1 2 2 2 1 1 2 2 24594 106 4 4 4 4 4 4 4 4 4 4 001 /07 24641 2 21 1 2 2 2 1 1 2 2 001 /07 24643 2 2 1 1 2 2 2 1 1 2 2 001 /07 24646 2 21 1 2 2 2 1 1.3 2 2 ATCC 7000057 QC 2 1 1 2 2 2 1.7 1.7 2 2 ATCC 29213SA QC 2 1.3 2 2 Capsules Reference Standard RIBOTYPE Fresh 14d 30d FreshRMA# RF REFRIG RF REFRIG 24608 002 2 1 1 2 2 1 24605 002 2 1 1 2 2 124655 005 2 2 1.3 2 2 2 24615 005 2.7 2.7 2 2.7 2 2 24678 012 4 4 4 4 44 24628 012 2 1 1 2 2 1 24527 014 1.7 1 1 2 1.3 1 24529 014 2 1 1 2 1.31 24547 014 2 1 1 2 1 1 24541 020 1.7 1 1 2 1.7 1 24543 020 1.7 1 1 21.7 1 24567 020 2 1 1 2 1.7 1 24504 027 2 1 1 2 2 1 24510 027 2 1 1 2 21 24519 027 2 1 1 2 1.3 1 24683 027 2 2 2 2 1.7 2 24690 027 4 4 4 4 4 424561 078 1 1 1 1 1 1 24575 078 1.3 1 1 2 1 1 24601 106 2 1.7 1 1.7 2 224607 106 2 1 1 2 2 1 24594 106 4 4 4 4 4 4 001 /07 24641 2 2 1 1 2 2 1001 /07 24643 2 2 1 1 2 2 1 001 /07 24646 2 2 1 1 2 2 1 ATCC 7000057 QC2 1.7 1.3 2 2 1 C. difficile ATCC 29213 SA CQ 2 1.3 2 S. aureus

TABLE 3 Average MIC Solution of Invention IV Capsules Fresh 1.9 2.2 2.214 RT 1.5 1.5 1.5 14 REF 1.4 1.5 1.5 30 RT 2.2 2.2 2.2 30 REF 1.8 2.1 2

Example 9: In Vitro Study of the Activity of Two Different Preparationsof Vancomycin (Sigma, Cutis) Against Clostridium difficile With KnownRibotypes

Introduction: The purpose of the study was to compare the MICs obtainedwith the formulation of vancomycin HCL of the invention (Cutis) toformulations obtained using a vancomycin HCL reference standard powderobtained from Sigma. The Exmaple also involves the analysis of asolution or diluent to determine if it has antimicrobial activity at theconcentrations present in the agar dilution test.

Materials

-   Brucella agar deeps - Anaerobe systems, Lot 3021809-   Vancomycin HCl, Sigma, Lot SLBG4650V-   Vancomycin, Cutis, Lot E951-   Na citrate, Baker, Lot 423343-   Na benzoate, Emerald, Lot KASBD46212

Methods: The C. difficile were tested by agar dilution according to theprocedures in the Clinical and Laboratory Standards Institute. 2012.Methods for antimicrobial susceptibility testing of anaerobic bacteria;approved standard-seventh edition.

Other anaerobic organisms included clinical isolates of Bifidobacteriumadolescentis and Bacteroides fragilis. Clostridium difficile ATCC 700057was included as a quality control strain.

The C. difficile isolates were recovered from patients with CDI duringthe past two years and stored at -70° C. in 20% skim milk. Ribotypingwas performed by another reference laboratory. The strains were takenfrom the freezer and subcultured at least twice to ensure purity andgood growth.

On the day of testing, they were suspended in Brucella broth to theturbidity of the 0.5 McFarland standard for testing.

Stock solution of the Sigma vancomycin was prepared in water and storedat -70° C. On the day of testing, it was thawed and further diluted inwater to obtain the desired range of 16 -0.125 µg/ml. The Cutisvancomycin (25 mg/ml) was reconstituted on the day of testing andfurther diluted for a final range of 16-0.125 µg/ml in the test. Theantimicrobials were added to molten Brucella agar supplemented withvitamin K and hemin to prepare the plates. Drug-free growth controlswere included for testing before and after each set of plates. Alltesting was done in triplicate.

The organisms were pipetted into the wells of the Steers replicator headand applied to the plates with the multi-pronged replicator. The finalconcentration of organisms was ~10⁵ CFU/spot.

The plates were incubated in the anaerobic chamber incubator for 44h andexamined for growth. The MIC is defined as the concentration of drugthat completely inhibits growth or results in a marked reduction ofgrowth compared to the drug-free growth control.

The results for the vancomycin MICs and the Na benzoate, Na citrate, andthe Cutis diluent are presented in Tables 4 and 5. The Cutis diluentcomprises: water, about 0.12% (w/v) citric acid (anhydrous), 0.2% (w/v)sucralose, about 0.05% (w/v) artificial grape flavor, 0.1% (w/v) sodiumbenzoate, about 0.0002% (w/v) D&C Yellow No. 10 and about 0.000038%(w/v) FD&C Red No. 40.

Results

TABLE 4 In vitro activity (ug/ml) of different preparations ofvancomycin ( Sigma, Cutis) against Clostridium difficile and otherenteric organisms tested by the agar dilution method Sigma Cutis S- VAN-S- VAN- S-VAN- C- VAN- C- VAN- C-VAN- RMA# organism Ribotype 1 2 3 AV 12 3 AV 24504 C. difficile 027 2 2 2 2 2 2 2 2 24510 C. difficile 027 2 21 1.7 2 2 2 2 24519 C. difficile 027 2 2 1 1.7 1 1 2 1.3 24527 C.difficile 014 2 2 2 2 1 2 1 1.3 24529 C. difficile 014 2 2 2 2 1 2 1 1.324541 C. difficile 020 1 2 2 1.7 2 2 2 2 24543 C. difficile 020 1 1 21.3 1 2 2 1.7 24547 C. difficile 014 1 1 1 1 2 2 2 2 24560 C. difficile078 2 2 1 1.7 2 1 2 1.7 24561 C. difficile 078 2 2 2 2 1 1 2 1.3 24567C. difficile 020 2 2 2 2 2 2 2 2 24575 C. difficile 078 2 4 2 2.7 1 1 11 24594 C. difficile 106 4 2 4 3.3 4 4 4 4 24601 C. difficile 106 2 2 22 2 2 2 2 24605 C. difficile 002 2 2 2 2 2 2 2 2 24607 C. difficile 1062 2 2 2 2 2 2 2 24608 C. difficile 002 2 2 2 2 2 2 2 2 24615 C.difficile 005 2 2 2 2 2 2 2 2 24628 C. difficile 012 2 2 2 2 2 2 2 224641 C. difficile 001/072 2 2 2 2 2 2 2 2 24643 C. difficile 001/072 22 2 2 2 2 2 2 24646 C. difficile 001/072 2 2 2 2 2 2 2 2 24655 C.difficile 005 2 2 2 2 2 2 4 2.7 24678 C. difficile 012 4 4 4 4 4 4 4 424683 C. difficile 027 2 2 2 2 2 2 2 2 24690 C. difficile 027 4 4 8 5.34 4 4 4 21317 Bif. adolescentis 1 1 1 1 1 1 1 1 21531 Bif. adolescentis0.5 1 1 0.83 1 1 1 1 21551 Bif. adolescentis 1 1 1 1 1 1 1 1 21680 Bif.adolescentis 1 1 1 1 1 1 1 1 21703 Bif. adolescentis 1 1 1 1 1 1 1 1ATCC 2 2 2 2 2 2 2 2 700057 26381 C. difficile 017 2 2 1 1.7 2 2 2 226442 C. difficile 017 1 1 1 1 1 1 1 1 26443 C. difficile 027 4 4 4 4 44 4 4 26445 C. difficile 017 2 2 2 2 2 2 2 2 26446 C. difficile 027 1 11 1 2 1 1 1.3 26447 C. difficile 001 2 2 2 2 1 2 2 1.7 26451 C.difficile 001 2 2 1 1.7 1 2 1 1.3 26455 C. difficile 027 1 1 1 1 1 1 1 126457 C. difficile 027 1 1 1 1 1 1 1 1 26461 C. difficile 106 1 2 1 1.32 1 1 1.3 26463 C. difficile 002 1 2 1 1.3 1 2 1 1.3 26466 C. difficile020 2 2 1 1.7 1 2 1 1.3 26467 C. difficile 002 1 2 1 1.3 1 2 1 1.3 26471C. difficile 001 2 2 2 2 2 2 2 2 26472 C. difficile 014 1 1 1 1 1 1 21.3 26480 C. difficile 106 1 1 1 1 2 1 2 1.7 26484 C. difficile 106 1 11 1 2 1 1 1.3 26485 C. difficile 078 2 1 1 1.3 2 1 1 1.3 26489 C.difficile 176 1 1 1 1 1 1 1 1 26494 C. difficile 126 1 2 1 1.3 1 2 1 1.326500 C. difficile 014 1 1 1 1 1 2 2 1.7 26503 C. difficile 014 1 1 1 12 1 1 1.3 26543 C. difficile 078 1 1 1 1 1 1 1 1 26544 C. difficile 0782 2 2 2 2 2 2 2 26563 C. difficile 018 1 1 1 1 1 1 1 1 26572 C.difficile 017 2 2 1 1.7 2 2 1 1.7 26982 B.fragilis >16 >16 >16 >16 >16 >16 >16 >16 26981 B.fragilis >16 >16 >16 >16 >16 >16 >16 >16 26980 B.fragilis >16 >16 >16 >16 >16 >16 >16 >16 26979 B.fragilis >16 >16 >16 >16 >16 >16 >16 >16 26978 B. fragilis >16 > 16 >16 > 16 > 16 > 16 > 16 > 16 2 2 2 2 2 ATCC 2 2 2 700057 C. difficile

AV, Average

TABLE 5 In vitro activity of diluent, Na benzoate and Na citrate, andagainst Clostridium difficile and other enteric organisms tested by theagar dilution method diluent diluent Na benz 0.02 Na benz 0.01 Na benz0.00 Na RMA# organism Ribotype 10% 20% % % 5% citrate 24504 C. difficile027 gr gr gr gr gr > 0.5% 24510 C. difficile 027 gr gr gr gr gr > 0.5%24519 C. difficile 027 gr gr gr gr gr > 0.5% 24527 C. difficile 014 grgr gr gr gr > 0.5% 24529 C. difficile 014 gr gr gr gr gr > 0.5% 24541 C.difficile 020 gr gr gr gr gr > 0.5% 24543 C. difficile 020 gr gr gr grgr > 0.5% 24547 C. difficile 014 gr gr gr gr gr > 0.5% 24560 C.difficile 078 gr gr gr gr gr > 0.5% 24561 C. difficile 078 gr gr gr grgr > 0.5% 24567 C. difficile 020 gr gr gr gr gr > 0.5% 24575 C.difficile 078 gr gr gr gr gr > 0.5% 24594 C. difficile 106 gr gr gr grgr > 0.5% 24601 C. difficile 106 gr gr gr gr gr > 0.5% 24605 C.difficile 002 gr gr gr gr gr > 0.5% 24607 C. difficile 106 gr gr gr grgr > 0.5% 24608 C. difficile 002 gr gr gr gr gr > 0.5% 24615 C.difficile 005 gr gr gr gr gr > 0.5% 24628 C. difficile 012 gr gr gr grgr > 0.5% 24641 C. difficile 001/072 gr gr gr gr gr > 0.5% 24643 C.difficile 001/072 gr gr gr gr gr > 0.5% 24646 C. difficile 001/072 gr grgr gr gr > 0.5% 24655 C. difficile 005 gr gr gr gr gr > 0.5% 24678 C.difficile 012 gr gr gr gr gr > 0.5% 24683 C. difficile 027 gr gr gr grgr > 0.5% 24690 C. difficile 027 gr gr gr gr gr > 0.5% 700057 C.difficile QC gr gr gr gr gr > 0.5% 26381 C. difficile 017 gr gr gr grgr > 0.5% 26442 C. difficile 017 gr gr gr gr gr > 0.5% 26443 C.difficile 027 gr gr gr gr gr > 0.5% 26445 C. difficile 017 gr gr gr grgr > 0.5% 26446 C. difficile 027 gr gr gr gr gr > 0.5% 26447 C.difficile 001 gr gr gr gr gr > 0.5% 26451 C. difficile 001 gr gr gr grgr > 0.5% 26455 C. difficile 027 gr gr gr gr gr > 0.5% 26457 C.difficile 027 gr gr gr gr gr > 0.5% 26461 C. difficile 106 gr gr gr grgr > 0.5% 26463 C. difficile 002 gr gr gr gr gr > 0.5% 26466 C.difficile 020 gr gr gr gr gr > 0.5% 26467 C. difficile 002 gr gr gr grgr > 0.5% 26471 C. difficile 001 gr gr gr gr gr > 0.5% 26472 C.difficile 014 gr gr gr gr gr > 0.5% 26480 C. difficile 106 gr gr gr grgr > 0.5% 26484 C. difficile 106 gr gr gr gr gr > 0.5% 26485 C.difficile 078 gr gr gr gr gr > 0.5% 26489 C. difficile 176 gr gr gr grgr > 0.5% 26494 C. difficile 126 gr gr gr gr gr > 0.5% 26500 C.difficile 014 gr gr gr gr gr > 0.5% 26503 C. difficile 014 gr gr gr grgr > 0.5% 26543 C. difficile 078 gr gr gr gr gr > 0.5% 26544 C.difficile 078 gr gr gr gr gr > 0.5% 26563 C. difficile 018 gr gr gr grgr > 0.5% 26572 C. difficile 017 gr gr gr gr gr > 0.5% 21317 Bif.adolescentis gr gr gr gr gr > 0.5% 21531 Bif. adolescentis gr gr gr grgr > 0.5% 21551 Bif. adolescentis gr gr gr gr gr > 0.5% 21680 Bif.adolescentis gr gr gr gr gr > 0.5% 21703 Bif. adolescentis gr gr gr grgr > 0.5% 26982 B. fragilis gr gr gr gr gr > 0.5% 26981 B. fragilis grgr gr gr gr > 0.5% 26980 B. fragilis gr gr gr gr gr > 0.5% 26979 B.fragilis gr gr gr gr gr > 0.5% 26978 B. fragilis gr gr gr gr gr > 0.5%700057 C. difficile gr gr gr gr gr > 0.5% gc, growth control ng, nogrowth gr, growth

Discussion: The vancomycin MICs for both preparations were very similarwith both producing mostly MICs of 1 or 2 µg/ml with a few isolates at 4µg/ml. The triplicate MICs were averaged and are shown in a separatecolumn. The diluent at the concentration present in the test (~0.01%)did not show inhibition, and when tested at much higher concentrationsof 10% or 20%, also did not show any growth inhibition for any of thestrains. Since two of the ingredients in the diluent are Na benzoate andNa citrate, we tested those separately and also saw no inhibition atconcentrations exceeding those present in the diluent.

Cutis vancomycin solution is comparable to the standard vancomycin asmeasured by the agar dilution method. The diluent tested at much higherconcentrations (100X- 200X) than present in the dilution series showedno antimicrobial activity.

Example 10: In Vitro Activity of Different Preparations of Vancomycin,Na Benzoate, and Diluent Against Staphylococcus aureus and Other EntericOrganisms

Diluent was mixed 50:50 with MH broth; additional growth control was MHbroth mixed 50:50 with water. While Klebsiella pneumonia, and E. coligrew in the 50% diluent, the buttons were about 50% the diameter of thegrowth control, suggesting that some inhibition was occurring.

Surprisingly the Cutis diluent or liquid solution of the invention wasable to effectively inhibit mold and yeast growth for long periods oftime at room temperature without impacting the growth of C. diff.

RMA# organism S- VAN S- VAN S- VAN C- VAN C- VAN C- VAN diluent 50% GC50% Na benz Na benz 0.02% 0.01% 18546 S. aureus 1 1 1 ng ng ng ng gr grgr 18547 S. aureus 2 1 1 ng ng ng ng gr gr gr 18683 S. aureus 0.5 0.50.5 ng ng ng ng gr gr gr 18913 S. aureus 1 0.5 0.5 ng ng ng ng gr gr gr19065 S. aureus 1 1 1 ng ng ng ng gr gr gr 19066 S. aureus 0.5 0.5 0.5ng ng ng ng gr gr gr 19831 S. aureus 0.5 0.5 0.5 ng ng ng ng gr gr gr19841 S. aureus 1 1 1 ng ng ng ng gr gr gr 21291 S. aureus 0.5 0.5 0.5ng ng ng ng gr gr gr 21743 S. aureus 0.5 0.5 0.5 ng ng ng ng gr gr gr22670 S. aureus 1 0.5 0.5 ng ng ng ng gr gr gr 22727 S. aureus 0.5 0.50.5 ng ng ng ng gr gr gr 22738 S. aureus 0.5 0.5 0.5 ng ng ng ng gr grgr 25524 S. aureus 1 0.5 1 ng ng ng ng gr gr gr 22932 S. aureus 2 1 1 ngng ng ng gr gr gr 22934 S. aureus 1 1 1 ng ng ng ng gr gr gr 22935 S.aureus 1 1 1 ng ng ng ng gr gr gr 23270 S. aureus 1 1 1 ng ng ng ng grgr gr 23271 S. aureus 1 1 1 ng ng ng ng gr gr gr 23272 S. aureus 0.5 0.50.5 ng ng ng ng gr gr gr 23350 S. aureus 1 1 1 ng ng ng ng gr gr gr23351 S. aureus 1 1 1 ng ng ng ng gr gr gr 23352 S. aureus 1 0.5 1 ng ngng ng gr gr gr 23394 S. aureus 0.5 0.5 0.5 ng ng ng ng gr gr gr 23395 S.aureus 1 0.5 1 ng ng ng ng gr gr gr 23396 S. aureus 0.5 0.5 0.5 ng ng ngng gr gr gr 23397 S. aureus 0.5 0.5 0.5 ng ng ng ng gr gr gr 23398 S.aureus 1 1 0.5 ng ng ng ng gr gr gr 23399 S. aureus 1 1 1 ng ng ng ng grgr gr 23401 S. aureus 0.5 0.5 0.5 ng ng ng ng gr gr gr 23502 S. aureus 10.5 1 ng ng ng ng gr gr gr 23509 S. aureus ng ng ng ng gr gr gr 23613 S.aureus 1 1 0.5 ng ng ng ng gr gr gr 23614 S. aureus 1 1 1 ng ng ng ng grgr gr 23615 S. aureus 0.5 0.5 0.5 ng ng ng ng gr gr gr 23616 S. aureus0.5 0.5 0.5 ng ng ng ng gr gr gr 23617 S. aureus 0.5 0.5 0.5 ng ng ng nggr gr gr 23618 S. aureus 1 0.5 1 ng ng ng ng gr gr gr 23619 S. aureus0.5 0.5 0.5 ng ng ng ng gr gr gr 23620 S. aureus 0.5 0.5 0.5 ng ng ng nggr gr gr 23621 S. aureus 1 1 1 ng ng ng ng gr gr gr 23622 S. aureus 1 11 ng ng ng ng gr gr gr 23623 S. aureus 1 1 1 ng ng ng ng gr gr gr 23812S. aureus 1 1 1 ng ng ng ng gr gr gr 24160 S. aureus 1 1 1 ng ng ng nggr gr gr 24161 S. aureus 1 1 1 ng ng ng ng gr gr gr 24162 S. aureus 0.50.5 0.5 ng ng ng ng gr gr gr 24163 S. aureus 0.5 0.5 0.5 ng ng ng ng grgr gr 24164 S. aureus 1 0.5 0.5 ng ng ng ng gr gr gr 24463 S. aureus 0.50.5 0.5 ng ng ng ng gr gr gr ATCC S. aureus 2 2 2 ng ng ng ng gr gr grATCC S. aureus 2 1 1 ng ng ng ng gr gr gr

Example 11: Stability Studies Have Indicated That Vancomycin HClSolution Is Stable When Stored in oral Syringes. The Study Was Performedon the 25 Mg/Ml Vancomycin Solution in Clear and Amber Oral Syringes.The Tests Included Appearance, pH, and Microbial Assay (USP <81>). TheResults Indicate That Vancomycin HCl Solution When Stored at BothRefrigerated (2 -8° C.) and Ambient (25° C./60%RH) Conditions Are Stablefor at Least 3 Days

Vancomycin HCl Stability in Oral Syringe Results Sample Storage TimePoints Initial Day 3 Average Antibiotics Microbial Assay (%)Specification: 90.0 to 115.0 % pH Specific ation: pH 2.5-4.5 Appear anceAverage Antibiotics Microbial Assay (%) Specifica tion: 90.0 to 115.0%pH Specificat ion: pH 2.5-4.5 Appeara nce Vancom ycin Clear Syringe 2 -8° C. 102.2 3.49 pale yellow liquid 101.3 3.15 pale yellow liquid 25°C./60 %RH 101.2 3.47 pale yellow liquid 97.9 3.16 pale yellow liquidVancom ycin Amber Syringe 2 - 8° C. 104.4 3.42 pale yellow liquid 102.23.12 pale yellow liquid 25° C./60 %RH 103.8 3.40 pale yellow liquid102.0 3.13 pale yellow liquid

Example 12: Chemical Analysis at 30 Days: Stability Studies HaveIndicated That Vancomycin HCl Solution Is Stable for at Least 30 DaysWhen Stored in HDPE Container-Closure System When Tested Using aChemical Analysis to Assay Vancomycin Content. The Study Was Performedon the 25 mg/mL Solution. The Results Indicate That Vancomycin HClSolution When Stored at Refrigerated (2 - 8° C.) Conditions Is Stablefor at Least 30 Days

Vancomycin HCl Stability (Chemical Analysis to Assay Vancomycin Content)Sample Storage Time Points Initial Day 30 Vancomycin AssaySpecification: 90.0-110.0% Vancomycin Assay Specification: 90.0-110.0%Vancomy cin 25 mg/ml 2 - 8° C. 97.7% 96.9%

Example 13: Dissolution Studies for Three Commercially AvailableProducts: Vancomycin (i.e. Vancomycin Oral Liquid of the Invention,Vancomycin Hydrochloride Powder for Injection, and Vancocin® Capsules)

The studies showed that both vancomycin oral liquid of the invention andvancomycin hydrochloride powder for injection demonstrate the samedissolution profile and are both considered rapidly dissolving (i.e. atleast 85% dissolution in 15 mins.; see FIGS. 3 to 6 ). Vancocin®capsules show a slower dissolution profile compared with the twosolution forms (see FIGS. 3 to 6 ). The dissolution tests was performedwith Apparatus II (rotating paddle, 100 rpm) using four media, water,buffer pH 4.5, buffer pH 6.8, and 0.1N HCl and were sampled at 10, 15,20, 30, 45, and 60 minutes. The dissolution profiles show that bothvancomycin oral liquid of the invention and vancomycin hydrochloridepowder for injection are more readily available compared with Vancocin®capsules in all four media.

Example 14: Appearance of Lyophilized Vancomycin Hydrochloride Powder inSterile Water for Injection Compared to the Vancomycin Oral Liquid ofthe Invention at Ambient and Refrigerated Temperature

Vancomycin STERILE WATER Vancomycin SOLUTION OF THE INVENTION China(Medisca) China (Medisca) Lot 111058/A Lot 111058/A 3.84 g/147ml STERILWATER (Lot 41-326-DK Hospira Inc.) 3.84 g/147 ml SOLUTION (Lot NWA004B)2-8C RT 2-8C RT Appearance pH Precipitate Appearance pH PrecipitateAppearance pH Precipitate Appearance pH Precipitate Days Time 0 1/26/15Light Yellow 2.9 1 Light Yellow 2.8 1 Clear Golden Yellow to Amber 2.9 1Clear Golden Yellow to Amber 2.9 1 1 Snow day 2 1/28/15 Light Yellow 2.91 Light Yellow 3.0 1 Clear Golden Yellow to Amber 3.1 1 Clear GoldenYellow to Amber 3.0 1 3 1/29/15 Light Yellow 2.8 1 Light Yellow 3.0 1Clear Golden Yellow to Amber 2.9 1 Clear Golden Yellow to Amber 3.0 1 41/30/15 Light Yellow 2.9 1 Light Yellow 2.8 1 Clear Golden Yellow toAmber 3.0 1 Clear Golden Yellow to Amber 3.1 1 5 1/31/15 Weekend WeekendWeekend Weekend 6 2/1/15 Weekend Weekend Weekend Weekend 7 2/2/15 Snowday Snow day Snow day Snow day 8 2/3/15 Light Yellow 2.7 1 Light Yellow2.7 1 Clear Golden Yellow to Amber 2.9 1 Clear Golden Yellow to Amber2.8 1 15 2/10/15 Light Yellow 2.9 1 Light Yellow 2.9 1 Clear GoldenYellow to Amber 2.9 1 Clear Golden Yellow to Amber 2.9 1 21 2/17/15Light Yellow 2.8 1 Light Yellow 2.7 1 Clear Golden Yellow to Amber 2.8 1Clear Golden Yellow to Amber 2.9 1 28 2/23/15 Light Yellow 2.9 1 LightYellow 2.8 1 Clear Golden Yellow to Amber 2.9 1 Clear Golden Yellow toAmber 3.0 1 35 3/2/15 Light Yellow Light Yellow 36 3/3/15 Light Yellow 1Light Yellow 1 Clear Golden Yellow to Amber 1 Clear Golden Yellow toAmber 1 37 3/4/15 Light Yellow 1 Light Yellow 1 Clear Golden Yellow toAmber 1 Clear Golden Yellow to Amber 1 38 3/5/15 Light Yellow 1 LightYellow 1 Clear Golden Yellow to Amber 1 Clear Golden Yellow to Amber 139 3/6/15 Light Yellow 1 Light Yellow 1 Clear Golden Yellow to Amber 1Clear Golden Yellow to Amber 1 42 3/9/15 Light Yellow 1 Light Yellow 1Clear Golden Yellow to Amber 1 Clear Golden Yellow to Amber 1 1=Free ofparticles 2=slightly hazy 3=small amount of particles visible 4=largeamount of particles visible 5=completely out of solution VancomycinSTERILE WATER Vancomycin SOLUTION OF THE INVENTION Hungary (Medisca)Hungary (Medisca) Lot B1231901 Lot B1231901 3.84 g/147ml STERIL WATER(Lot 41-326-DK Hospira Inc.) 3.84 g/147 ml SOLUTION (Lot NWA004B) 2-8CRT 2-8C RT Appearance pH Precipitate Appearance pH PrecipitateAppearance pH Precipitate Appearance pH Precipitate Days Time 0 1/26/15Clear 3.5 1 Clear 3.4 1 Clear Golden Yellow to Amber 3.3 1 Clear GoldenYellow to Amber 3.3 1 1 Snow day 2 1/28/15 Clear 3.6 1 Clear 3.6 1 ClearGolden Yellow to Amber 3.4 1 Clear Golden Yellow to Amber 3.5 1 31/29/15 Clear 3.6 1 Clear 3.6 1 Clear Golden Yellow to Amber 3.5 1 ClearGolden Yellow to Amber 3.5 1 4 1/30/15 Clear 3.3 1 Clear 3.5 1 ClearGolden Yellow to Amber 3.5 1 Clear Golden Yellow to Amber 3.5 1 51/31/15 Weekend Weekend Weekend Weekend 6 2/1/15 Weekend Weekend WeekendWeekend 7 2/2/15 Snow day Snow day Snow day Snow day 8 2/3/15 Clear 3.41 Clear 3.5 1 Clear Golden Yellow to Amber 3.2 1 Clear Golden Yellow toAmber 3.2 1 15 2/10/15 Clear 3.4 1 Clear 3.6 1 Clear Golden Yellow toAmber 3.3 1 Clear Golden Yellow to Amber 3.5 1 21 2/17/15 Clear 3.6 1Clear 3.3 1 Clear Golden Yellow to Amber 3.2 1 Clear Golden Yellow toAmber 3.3 1 28 2/23/15 Clear 3.5 1 Clear 3.9 1 Clear Golden Yellow toAmber 3.3 1 Clear Golden Yellow to Amber 3.4 1 35 3/2/15 Clear 1 2 1Clear Golden Yellow to Amber 1 36 3/3/15 Clear 1 3 Clear Golden Yellowto Amber 1 Clear Golden Yellow to Amber 1 37 3/4/15 Clear 1 3 ClearGolden Yellow to Amber 1 Clear Golden Yellow to Amber 1 38 3/5/15 Clear1 4 Clear Golden Yellow to Amber 1 Clear Golden Yellow to Amber 1 393/6/15 Clear 1 4 Clear Golden Yellow to Amber 1 Clear Golden Yellow toAmber 1 42 3/9/15 Clear 1 5 Clear Golden Yellow to Amber 1 Clear GoldenYellow to Amber 1 Vancomycin STERILE WATER Vancomycin SOLUTION OF THEINVENTION Denmark (Xellia) Denmark (Xellia) Lot A3330040 Lot A33300403.84 g/147 ml STERIL WATER (Lot 41-326-DK Hospira Inc.) 3.84 g/147 mlSOLUTION (Lot NWA004B) 2-8C RT 2-8C RT Appearance pH PrecipitateAppearance pH Precipitate Appearance pH Precipitate Appearance pHPrecipitate Days Time 0 1/26/15 Light Pink 3.8 1 Light Pink 3.2 1 ClearGolden Yellow to Amber 3.4 1 Clear Golden Yellow to Amber 3.3 1 1 Snowday 2 1/28/15 Light Pink 3.4 1 Light Pink 3.5 1 Clear Golden Yellow toAmber 3.4 1 Clear Golden Yellow to Amber 3.4 1 3 1/29/15 Light Pink 3.31 Light Pink 3.5 1 Clear Golden Yellow to Amber 3.4 1 Clear GoldenYellow to Amber 3.4 1 4 1/30/15 5 1/31/15 Weekend Weekend WeekendWeekend 6 2/1/15 Weekend Weekend Weekend Weekend 7 2/2/15 Snow day Snowday Snow day Snow day 8 2/3/15 Light Pink 3.2 1 Light Pink 3.2 1 ClearGolden Yellow to Amber 3.2 1 Clear Golden Yellow to Amber 3.2 1 152/10/15 Light Pink 3.3 1 Light Pink 3.4 1 Clear Golden Yellow to Amber3.3 1 Clear Golden Yellow to Amber 3.3 1 21 2/17/15 Light Pink 3.2 1Light Pink 3.1 1 Clear Golden Yellow to Amber 3.1 1 Clear Golden Yellowto Amber 3.2 1 28 2/23/15 Light Pink 3.1 1 Light Pink 3.5 1 Clear GoldenYellow to Amber 3.2 1 Clear Golden Yellow to Amber 3.4 1 35 3/2/15 LightPink 1 1 36 3/3/15 Light Pink 1 1 Clear Golden Yellow to Amber 1 ClearGolden Yellow to Amber 1 37 3/4/15 Light Pink 1 2 Clear Golden Yellow toAmber 1 Clear Golden Yellow to Amber 1 38 3/5/15 Light Pink 1 3 ClearGolden Yellow to Amber 1 Clear Golden Yellow to Amber 1 39 3/6/15 LightPink 1 3 Clear Golden Yellow to Amber 1 Clear Golden Yellow to Amber 142 3/9/15 Light Pink 1 5 Clear Golden Yellow to Amber 1 Clear GoldenYellow to Amber 1

Example 15: Caco-2 Permeability Study

Caco-2 cells are used as an in vitro model of the human intestinalepithelium and permit assessment of the intestinal permeability ofpotential drugs. Vancomycin is added to either the apical or basolateralside of a confluent monolayer of Caco-2 cells and permeability ismeasured by monitoring the appearance of the Vancomycin on the oppositeside of the monolayer using LC-MS/MS. When performing a bi-directionalassay, the efflux ratio (ER) is calculated from the ratio of B->A andA->B permeabilities.

The permeability measure of vancomycin in the apical to basolateral(A->B) and basolateral to apical (B->A) direction across Caco-2 cells,and to determine the efflux ratio. Three different commerciallyavailable products of vancomycin were tested at a pH of 5.7, 6.5, and7.4: vancomycin oral liquid of the invention, lyophilized vancomycinhydrochloride powder for injection in solution, and vancomycinhydrochloride capsules. Listed in the table below are the preliminarydata for the Papp rate coefficients of these test articles (at theindicated pH values).

Test Article mean A->B Papp (x10-6 cm s-1) mean B->A Papp (x10-6 cm s-1)Ranitidine 0.3 1.5 Talinolol 0.1 10.2 Metoprolol 11.6 30.0 VancomycinOral Liquid of the Invention @ pH5.7 <0.3 <0.3 Vancomycin Injection @pH5.7 <0.3 <0.3 Vancomycin Capsule @ pH5.7 <0.3 <0.3 Vancomycin OralLiquid of the Invention @ pH6.5 <0.3 <0.3 Vancomycin Injection @ pH6.5<0.3 <0.3 Vancomycin Capsule @ pH6.5 <0.3 <0.3 Vancomycin Oral Liquid ofthe Invention @ pH7.4 <0.3 <0.3 Vancomycin Injection @ pH7.4 <0.3 <0.3Vancomycin Capsule @ pH7.4 ≤0.86 <0.3

REFERENCES

U.S. Pat. 4,670,258 to Harris et al.

Fraser TG, Swiencicki JF. Clostridium difficile. In: Cleveland Clinic:Center for continuting education.

<http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/infectious-disease/clostridum-difficile-infection/>.Accessed Aug. 15, 2013.

Ryan KJ, Ray CG. Sherris Medical Microbiology (4th ed.). McGraw Hill.2004. 322-4.

Hall IC, O′Toole E. Intestinal flora in newborn infants with adescription of a new pathogenic anaerobe, Bacillus difficilis. Am J DisChild. 1935;49:390-402.

Baron EJ, Tenover FC. The emerging threat of Clostridium difficileinfection. <http://www.cepheid.com/media/files/brochures/CDiff>.Accessed Aug. 15, 2013.

Bartlett, JG. Narrative review: the new epidemic of Clostridiumdifficile-associated enteric disease. Ann Intern Med. 2006;145:758.

Weir E., Flegel K. Protecting against Clostridium difficile illness. CanMed Assoc J. 2005;172:1178.

Pawar D, Bhandari P, Allenby K. A Review: Clostridiumdifficile-Associated Diarrhea. Indian Medical Gazette.2011.

CDC. Sehulster L, Chinn RYW. Guidelines for environmental infectioncontrol in healthcare facilities. MMWR. 2003;52(RR10);1-42.

Hull MW, Beck PL. Clostridium difficile-associated colitis. Can FamPhysician. 2004;50:1536-40, 1543-5.

Bartlett, JG. The Case for Vancomycin as the Preferred Drug forTreatment of Clostridium difficile Infection. CID. 2008;46:1489-1492.

Johnson S. Homann SR, Bettin KM, et al. Treatment of asymptomaticClostridium difficile carriers (fecal excretors) with vancomycin ormetronidazole: a randomized, placebo-controlled trial. Ann Intern Med.1992;117:297-302.

Gennaro AR. Remington: The Science and Practice of Pharmacy. LippincottWilliams & Wilkins. 20^(th) Ed: 1018-1021. 2000.

Burdock GA. Fenaroli’s Handbook of Flavor Ingredients. Cleveland: CRC.1994.

U.S. Pharmacist. Vancomycin Survey Results. March 2013.

P\S\L Research. Pharmacist Compounding Study. October 2012.

Mallet L, Sesin GP, Ericson J, et al. Storage of vancomycin oralsolution. N Engl J Med. 1982; 307(7): 445.

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The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

What is claimed is:
 1. A non-sterile stable liquid formulationcomprising a compounded solution of vancomycin hydrochloride that ishomogenous and stable for at least 30 days at ambient and refrigeratedtemperature conditions, wherein the compounded solution is prepared fromvancomycin hydrochloride and a liquid solution that is not Ora-Sweet. 2.The liquid formulation of claim 1, wherein the liquid solution is stablefor up to 3 months at accelerated conditions.
 3. The liquid formulationof claim 1, wherein the liquid solution is stable for up to 24 months atambient conditions.
 4. The liquid formulation of claim 1, wherein theliquid solution comprises: (a) a buffering agent, (b) water, (c) asweetener, (d) a flavoring agent, (e) a preservative, and (f) a dye. 5.The liquid formulation of claim 4, wherein the buffering agent is about0.12% (w/v) citric acid (anhydrous).
 6. The liquid formulation of claim4, wherein the sweetener is about 0.2% (w/v) sucralose.
 7. The liquidformulation of claim 4, wherein the flavoring agent is about 0.05% (w/v)artificial grape flavor.
 8. The liquid formulation of claim 4, whereinthe preservative is about 0.1% (w/v) sodium benzoate.
 9. The liquidformulation of claim 4, wherein the dye comprises about 0.0002% (w/v)D&C Yellow No. 10 and about 0.000038% (w/v) FD&C Red No.
 40. 10. Theliquid formulation of claim 1, wherein the compounded solution isformulated for use in the treatment of C. difficile pseudomembranouscolitis and Staphylococcal enterocolitis.
 11. The liquid formulation ofclaim 1, wherein the compounded solution is formulated to inhibit thegrowth of bacteria, mold and yeast for at least 30 days at ambient andrefrigerated temperature conditions.
 12. The liquid formulation of claim1, wherein the liquid solution is formulated to inhibit the growth ofmold and yeast for greater than 30 days at room temperature conditions.13. The liquid formulation of claim 1, wherein the concentration ofvancomycin in the compounded solution is 1-100 mg/ml.
 14. The liquidformulation of claim 1, wherein the concentration of vancomycin in thecompounded solution is 20-60 mg/ml.
 15. The liquid formulation of claim1, wherein the concentration of vancomycin in the compounded solution is25-50 mg/ml. 16-19. (canceled)
 20. A non-sterile stable liquidformulation comprising a compounded solution of vancomycin hydrochloridethat is homogenous and stable for at least 30 days at ambient andrefrigerated temperature conditions, wherein the compounded solution hasa high solubility in water and a pH of about 2.5-4.5.
 21. The liquidformulation of claim 20, wherein the compounded solution is an ambercolor.
 22. The liquid formulation of claim 20, wherein the compoundedsolution includes a sweetener.
 23. A non-sterile stable liquidformulation comprising a compounded solution of vancomycin hydrochloridethat has been dissolved in a liquid solution comprising a preservative,wherein the compounded solution is stable for at least 30 days atambient and refrigerated temperature conditions and wherein thepreservative is present in the liquid solution at a concentration rangeof 0.08- 0.2% (w/v).
 24. A liquid solution comprising: (a) 0.1-0.4%(w/v) citric acid (anhydrous), (b) water, (c) 0.1-0.3% (w/v) sucralose,(d) 0.01-0.1 (w/v) of a flavoring agent, (e) 0.08- 0.2% (w/v) sodiumbenzoate, and (f) 0.0001-0.0003% (w/v) of a dye. 25-29. (canceled)